Antibacterial agents

ABSTRACT

A compound of formula (I) or a pharmaceutically or veterinarily acceptable salt thereof for the inhibition of activity of bacterial PDF enzyme: 
                         
wherein A represents a group of formula (IA) or (IB)
 
                         
and wherein R 1  is hydrogen, R 2  is n-butyl, benzyl or cyclopentylmethyl, R 3  is hydrogen, R 4  is tert-butyl, iso-butyl, benzyl or methyl, R 5  is hydrogen or methyl and R 6  is methyl.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 10/134,754, filed Apr. 30, 2002 now U.S. Pat. No. 6,787,522,entitled “Antibacterial Agents” which is hereby incorporated byreference in its entirety, which is a divisional application of U.S.application Ser. No. 09/355,489, filed Jan. 7, 2000, now U.S. Pat. No.6,423,690, issued Jul. 23, 2002, entitled “Antibacterial Agents” whichis hereby incorporated by reference in its entirety, which is a U.S.National Phase Patent Application of International Patent ApplicationPCT/GB99/00386 which claims priority to GB 9802549.7, filed Feb. 7,1998; GB 9806300.1 filed Mar. 24, 1998; GB 9810463.1 filed May 16, 1998;GB 9828318.7 filed Dec. 22, 1998 which are all incorporated by referencein their entirety.

FIELD OF THE INVENTION

This invention relates to the use of N-formyl hydroxylamine derivativesas antibacterial agents, to a novel class of such compounds, and topharmaceutical and veterinary compositions comprising such compounds.

BACKGROUND TO THE INVENTION

In general, bacterial pathogens are classified as either Gram-positiveor Gram-negative. Many antibacterial agents (including antibiotics) arespecific against one or other Gram-class of pathogens. Antibacterialagents effective against both Gram-positive and Gram-negative pathogensare therefore generally regarded as having broad spectrum activity.

Many classes of antibacterial agents are known, including thepenicillins and cephalosporins, tetracyclines, sulfonamides,monobactams, fluoroquinolones and quinolones, aminoglycosides,glycopeptides, macrolides, polymyxins, lincosamides, trimethoprim andchloramphenicol. The fundamental mechanisms of action of theseantibacterial classes vary.

Bacterial resistance to many known antibacterials is a growing problem.Accordingly there is a continuing need in the art for alternativeantibacterial agents, especially those which have mechanisms of actionfundamentally different from the known classes.

Amongst the Gram-positive pathogens, such as Staphylococci,Streptococci, Mycobacteria and Enterococci, resistant strains haveevolved/arisen which makes them particularly difficult to eradicate.Examples of such strains are methicillin resistant Staphylococcus aureus(MRSA), methicillin resistant coagulase negative Staphylococci (MRCNS),penicillin resistant Streptococcus pneumoniae and multiply resistantEnterococcus faecium.

Pathogenic bacteria are often resistant to the aminoglycoside, β-lactam(penicillins and cephalosporins), and chloramphenicol types ofantibiotic. This resistance involves the enzymatic inactivation of theantibiotic by hydrolysis or by formation of inactive derivatives. Theβ-lactam (penicillin and cephalosporin) family of antibiotics arecharacterised by the presence of a β-lactam ring structure. Resistanceto this family of antibiotics in clinical isolates is most commonly dueto the production of a “penicillinase” (β-lactamase) enzyme by theresistant bacterium which hydrolyses the β-lactam ring thus eliminatingits antibacterial activity.

Recently there has been an emergence of vancomycin-resistant strains ofenterococci (Woodford N. 1998 Glycopeptide-resistant enterococci: adecade of experience. Journal of Medical Microbiology. 47(10):849–62).Vancomycin-resistant enterococci are particularly hazardous in that theyare frequent causes of hospital based infections and are inherentlyresistant to most antibiotics. Vancomycin works by binding to theterminal D-Ala-D-Ala residues of the cell wall peptidioglycan precursor.The high-level resistance to vancomycin is known as VanA and isconferred by a genes located on a transposable element which alter theterminal residues to D-Ala-D-lac thus reducing the affinity forvancomycin.

In view of the rapid emergence of multidrug-resistant bacteria, thedevelopment of antibacterial agents with novel modes of action that areeffective against the growing number of resistant bacteria, particularlythe vancomycin resistant enterococci and β-lactam antibiotic-resistantbacteria, such as methicillin-resistant Staphylococcus aureus, is ofutmost importance.

BRIEF DESCRIPTION OF THE INVENTION

This invention is based on the finding that certain N-formylhydroxylamine derivatives have antibacterial activity, and makesavailable a new class of antibacterial agents. The inventors have foundthat the compounds with which this invention is concerned areantibacterial with respect to a range of Gram-positive and Gram-negativeorganisms. Furthermore, there is evidence that some compounds areantibacterial with respect to bacteria which are resistant to commonlyused antibiotics such as vancomycin and the β-lactam antibiotics, forexample methicillin-resistant Staphylococcus aureus.

Although it may be of interest to establish the mechanism of action ofthe compounds with which the invention is concerned, it is their abilityto inhibit bacterial growth which makes them useful. However, it ispresently believed that their antibacterial activity is due, at least inpart, to intracellular inhibition of bacterial polypeptide deformylase(PDF) enzyme.

Bacterial polypeptide deformylases (PDF) (EC 3.5.1.31), are a conservedfamily of metalloenzymes (Reviewed: Meinnel T, Lazennec C, Villoing S,Blanquet S, 1997, Journal of Molecular Biology 267, 749–761) which areessential for bacterial viability, their function being to remove theformyl group from the N-terminal methionine residue ofribosome-synthesised proteins in eubacteria. Mazel et al. (EMBO J.13(4):914–923, 1994) have recently cloned and characterised an E. coliPDF. As PDF is essential to the growth of bacteria and there is noeukaryotic counterpart to PDF, Mazel et al. (ibid), Rajagopalan et al.(J. Am. Chem. Soc. 119:12418–12419, 1997) and Becker et al., (J. BiolChem. 273(19):11413–11416, 1998) have each proposed that PDF is anexcellent anti-bacterial target.

Certain N-formyl hydroxylamine derivatives have previously been claimedin the patent publications listed below, although very few examples ofsuch compounds have been specifically made and described:

EP-B-0236872 (Roche) WO 92/09563 (Glycomed) WO 92/04735 (Syntex) WO95/19965 (Glycomed) WO 95/22966 (Sanofi Winthrop) WO 95/33709 (Roche) WO96/23791 (Syntex) WO 96/16027 (Syntex/Agouron) WO 97/03783 (BritishBiotech) WO 97/18207 (DuPont Merck) WO 98/38179 (GlaxoWellcome) WO98/47863 (Labs Jaques Logeais)

The pharmaceutical utility ascribed to the N-formyl hydroxylaminederivatives in those publications is the ability to inhibit matrixmetalloproteinases (MMPs) and in some cases release of tumour necrosisfactor (TNF), and hence the treatment of diseases or conditions mediatedby those enzymes, such as cancer and rheumatoid arthritis. That priorart does not disclose or imply that N-formyl hydroxylamine derivativeshave antibacterial activity.

In addition to these, U.S. Pat. No. 4,738,803 (Roques et al.) alsodiscloses N-formyl hydroxylamine derivatives, however, these compoundsare disclosed as enkephalinase inhibitors and are proposed for use asantidepressants and hypotensive agents. Also, WO 97/38705 (Bristol-MyersSquibb) discloses certain N-formyl hydroxylamine derivatives asenkephalinase and angiotensin converting enzyme inhibitors. This priorart does not disclose or imply that N-formyl hydroxylamine derivativeshave antibacterial activity either.

DTAILED DESCRIPTION OF THE INVENTION

According to the first aspect of the present invention there is providedthe use of a compound of formula (I) or a pharmaceutically orveterinarily acceptable salt thereof in the preparation of anantibacterial composition:

wherein:

-   R₁ represents hydrogen, or C₁–C₆ alkyl or C₁–C₆ alkyl substituted by    one or more halogen atoms;-   R₂ represents a group R₁₀—(X)_(n)-(ALK)_(m)— wherein    -   R₁₀ represents hydrogen, or a C₁–C₆ alkyl, C₂–C₆ alkenyl, C₂–C₆        alkynyl, cycloalkyl, aryl, or heterocyclyl group, any of which        may be unsubstituted or substituted by (C₁–C₆)alkyl,        (C₁–C₆)alkoxy, hydroxy, mercapto, (C₁–C₆)alkylthio, amino, halo        (including fluoro, chloro, bromo and iodo), trifluoromethyl,        cyano, nitro, —COOH, —CONH₂, —COOR^(A), —NHCOR^(A), —CONHR^(A),        —NHR^(A), —NR^(A)R^(B), or —CONR^(A)R^(B) wherein R^(A) and        R^(B) are independently a (C₁–C₆)alkyl group, and    -   ALK represents a straight or branched divalent C₁–C₆ alkylene,        C₂–C₆ alkenylene, or C₂–C₆ alkynylene radical, and may be        interrupted by one or more non-adjacent —NH—, —O— or —S—        linkages,    -   X represents —NH—, —O— or —S—, and    -   m and n are independently 0 or 1; and-   A represents (i) a group of formula (IA), (IB), (IC) or (ID)

wherein:

-   -   R₃ represents hydrogen and R₄ represents the side chain of a        natural or non-natural alpha amino acid or R₃ and R₄ when taken        together with the nitrogen and carbon atoms to which they are        respectively attached form an optionally substituted saturated        heterocyclic ring of 5 to 8 atoms which ring is optionally fused        to a carbocyclic or second heterocyclic ring,    -   R₅ and R₆, independently represent hydrogen, or optionally        substituted C₁–C₈ alkyl, cycloalkyl, aryl, aryl(C₁–C₆ alkyl),        heterocyclic, or heterocyclic(C₁–C₆ alkyl), or R₅ and R₆ when        taken together with the nitrogen atom to which they are attached        form an optionally substituted saturated heterocyclic ring of 3        to 8 atoms which ring is optionally fused to a carbocyclic or        second heterocyclic ring, and    -   R₇ represents hydrogen, C₁–C₆ alkyl, or an acyl group.

In another aspect, the invention provides a method for the treatment ofbacterial infections in humans and non-human mammals, which comprisesadministering to a subject suffering such infection an antibacteriallyeffective dose of a compound of formula (I) as defined above.

In a further aspect of the invention there is provided a method for thetreatment of bacterial contamination by applying an antibacteriallyeffective amount of a compound of formula (I) as defined above to thesite of contamination.

The compounds of formula (I) as defined above may be used ascomponent(s) of antibacterial cleaning or disinfecting materials.

According to a preferred embodiment, the various aspects of theinvention can be applied against vancomycin-, quinolone- and“β-lactam”-resistant bacteria and the infections they cause.

On the hypothesis that the compounds (I) act by inhibition ofintracellular PDF, the most potent antibacterial effect may be achievedby using compounds which efficiently pass through the bacterial cellwall. Thus, compounds which are highly active as inhibitors of PDF invitro and which penetrate bacterial cells are preferred for use inaccordance with the invention. It is to be expected that theantibacterial potency of compounds which are potent inhibitors of thePDF enzyme in vitro, but are poorly cell penetrant, may be improved bytheir use in the form of a prodrug, ie a structurally modified analoguewhich is converted to the parent molecule of formula (I), for example byenzymic action, after it has passed through the bacterial cell wall.

The invention also provides novel compounds of formula (I) above, orpharmaceutically or veterinarily acceptable salts thereof, wherein:

-   R₁ represents hydrogen, C₁–C₆ alkyl or C₁–C₆ alkyl substituted by    one or more halogen atoms;-   R₂ represents a group R₁₀—(ALK)_(m)— wherein    -   R₁₀ represents hydrogen, or a C₁–C₆ alkyl, C₂–C₆ alkenyl, C₂–C₆        alkynyl, a cycloalkyl, aryl, or heterocyclyl group, any of which        may be unsubstituted or substituted by (C₁–C₆)alkyl,        (C₁–C₆)alkoxy, hydroxy, mercapto, (C₁–C₆)alkylthio, amino, halo        (including fluoro, chloro, bromo and iodo), trifluoromethyl,        nitro, —COOH, —CONH₂, —COOR^(A), —NHCOR^(A), —CONHR^(A),        —NHR^(A), —NR^(A)R^(B), or —CONR^(A)R^(B) wherein R^(A) and        R^(B) are independently a (C₁–C₆)alkyl group,    -   ALK represents a straight or branched divalent C₁–C₆ alkylene,        C₂–C₆ alkenylene, C₂–C₆ alkynylene radical, and may be        interrupted by one or more non-adjacent —NH—, —O— or —S—        linkages, and    -   m represents 0 or 1;-   A represents a group of formula (IA), (IB), (IC) or (ID) above    wherein:    -   R₃ represents hydrogen and R₄ represents the side chain of a        natural or non-natural alpha amino acid or R₃ and R₄ when taken        together with the nitrogen and carbon atoms to which they are        respectively attached form an optionally substituted saturated        heterocyclic ring of 5 to 8 atoms which ring is optionally fused        to a carbocyclic or second heterocyclic ring,    -   R₅ and R₆, independently represent hydrogen, or optionally        substituted C₁–C₈ alkyl, cycloalkyl, aryl(C₁–C₆ alkyl),        non-aromatic heterocyclic, or heterocyclic(C₁–C₆ alkyl), or R₅        and R₆ when taken together with the nitrogen atom to which they        are attached form an optionally substituted saturated        heterocyclic ring of 3 to 8 atoms which ring is optionally fused        to a carbocyclic or second heterocyclic ring, and    -   R₇ represents hydrogen, C₁–C₆ alkyl, or an acyl group.

PROVIDED THAT (i) when A is a group of formula (IA) or (IB) and R₂ isC₂–C₅ alkyl then R₄ is not the side chain of a natural alpha amino acidor the side chain of a natural alpha-amino acid in which any functionalsubstituents are protected, any amino groups are acylated, and anycarboxyl groups are esterified;

-   -   (ii) when A is a group of formula (IA) or (IB) then R₄ is not a        bicyclicarylmethyl group; and    -   (iii) when A is a group of formula (IA) and R₂ is        cyclopropylmethyl, cyclobutylmethyl or cyclopentylmethyl and one        of R₅ and R₆ is hydrogen, then R₄ is not tert-butyl.

As used herein the term “(C₁–C₆)alkyl” means a straight or branchedchain alkyl moiety having from 1 to 6 carbon atoms, including forexample, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, t-butyl, n-pentyl and n-hexyl.

As used herein the term “divalent (C₁–C₆)alkylene radical” means asaturated hydrocarbon chain having from 1 to 6 carbon atoms and twounsatisfied valencies.

As used herein the term “(C₂–C₆)alkenyl” means a straight or branchedchain alkenyl moiety having from 2 to 6 carbon atoms having at least onedouble bond of either E or Z stereochemistry where applicable. The termincludes, for example, vinyl, allyl, 1- and 2-butenyl and2-methyl-2-propenyl.

As used herein the term “divalent (C₂–C₆)alkenylene radical” means ahydrocarbon chain having from 2 to 6 carbon atoms, at least one doublebond, and two unsatisfied valencies.

As used herein the term “C₂–C₆ alkynyl” refers to straight chain orbranched chain hydrocarbon groups having from two to six carbon atomsand having in addition one triple bond. This term would include forexample, ethynyl, 1-propynyl, 1- and 2-butynyl, 2-methyl-2-propynyl,2-pentynyl, 3-pentynyl, 4-pentynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl and5-hexynyl.

As used herein the term “divalent (C₂–C₆)alkynylene radical” means ahydrocarbon chain having from 2 to 6 carbon atoms, at least one triplebond, and two unsatisfied valencies.

As used herein the term “cycloalkyl” means a saturated alicyclic moietyhaving from 3–8 carbon atoms and includes, for example, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

As used herein the term “cycloalkenyl” means an unsaturated alicyclicmoiety having from 3–8 carbon atoms and includes, for example,cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyland cyclooctenyl. In the case of cycloalkenyl rings of from 5–8 carbonatoms, the ring may contain more than one double bond.

As used herein the term “aryl” refers to a mono-, bi- or tri-cycliccarbocyclic aromatic group, and to groups consisting of two covalentlylinked monocyclic carbocyclic aromatic groups. Illustrative of suchgroups are phenyl, biphenyl and napthyl.

As used herein the term “heteroaryl” refers to a 5- or 6-memberedaromatic ring containing one or more heteroatoms, and optionally fusedto a benzyl or pyridyl ring; and to groups consisting of two covalentlylinked 5- or 6-membered aromatic rings each containing one or moreheteroatoms; and to groups consisting of a monocyclic carbocyclicaromatic group covalently linked to a 5- or 6-membered aromatic ringscontaining one or more heteroatoms;. Illustrative of such groups arethienyl, furyl, pyrrolyl, imidazolyl, benzimidazolyl, thiazolyl,pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, thiadiazolyl,oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl,4-([1,2,3]-thiadiazoly-4-yl)phenyl and 5-isoxazol-3-ylthienyl.

As used herein the unqualified term “heterocyclyl” or “heterocyclic”includes “heteroaryl” as defined above, and in particular means a 5–7membered aromatic or non-aromatic heterocyclic ring containing one ormore heteroatoms selected from S, N and O, and optionally fused to abenzene ring, including for example, pyrrolyl, furyl, thienyl,piperidinyl, imidazolyl, oxazolyl, thiazolyl, thiadiazolyl, pyrazolyl,pyridinyl, pyrrolidinyl, pyrimidinyl, morpholinyl, piperazinyl, indolyl,benzimidazolyl, maleimido, succinimido, phthalimido and1,3-dioxo-1,3-dihydro-isoindol-2-yl groups.

As used herein the term “acyl” means a group R₂₀C(O)— where R₂₀ is(C₁–C₆)alkyl, (C₂–C₆)alkenyl, (C₃–C₇)cycloalkyl, phenyl, heterocyclyl,phenyl(C₁–C₆)alkyl, heterocyclyl(C₁–C₆)alkyl,(C₃–C₇)cycloalkyl(C₁–C₆)alkyl, phenyl(C₂–C₆)alkenyl,heterocyclyl(C₂–C₆)alkenyl, (C₃–C₇)cycloalkyl(C₂–C₆)alkenyl, any ofwhich R₂₀ groups may be substituted.

As used herein, the term “bicyclicarylmethyl” means (i) a methyl groupsubstituted by a monocyclic aryl or heteroaryl group which in turn issubstituted by a monocyclic aryl or heteroaryl group, or (ii) a methylgroup substituted by a monocyclic aryl or heteroaryl group to which isfused a second monocyclic aryl or heteroaryl group; and includes bothunsubstituted and substituted bicyclicarylmethyl. Examples of suchbicyclicarylmethyl groups include naphthyl, indolyl, quinolyl andisoquinolyl.

Unless otherwise specified in the context in which it occurs, the term“substituted” as applied to any moiety herein means substituted with upto four substituents, each of which independently may be (C₁–C₆)alkyl,benzyl, (C₁–C₆)alkoxy, phenoxy, hydroxy, mercapto, (C₁–C₆)alkylthio,amino, halo (including fluoro, chloro, bromo and iodo), trifluoromethyl,nitro, —COOH, —CONH₂, —COR^(A), —COOR^(A), —NHCOR^(A), —CONHR^(A),—NHR^(A), —NR^(A)R^(B), or —CONR^(A)R^(B) wherein R^(A) and R^(B) areindependently a (C₁–C₆)alkyl group. In the case where “substituted”means benzyl, the phenyl ring thereof may itself be substituted with anyof the foregoing, except benzyl.

As used herein the terms “side chain of a natural alpha-amino acid” and“side chain of a non-natural alpha-amino acid” mean the group R^(x) inrespectively a natural and non-natural amino acid of formulaNH₂—CH(R^(x))—COOH.

Examples of side chains of natural alpha amino acids include those ofalanine, arginine, asparagine, aspartic acid, cysteine, cystine,glutamic acid, histidine, 5-hydroxylysine, 4-hydroxyproline, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, valine, α-aminoadipic acid, α-amino-n-butyricacid, 3,4-dihydroxyphenylalanine, homoserine, α-methylserine, ornithine,pipecolic acid, and thyroxine.

In natural alpha-amino acid side chains which contain functionalsubstituents, for example amino, carboxyl, hydroxy, mercapto, guanidyl,imidazolyl, or indolyl groups as in arginine, lysine, glutamic acid,aspartic acid, tryptophan, histidine, serine, threonine, tyrosine, andcysteine, such functional substituents may optionally be protected.

Likewise, in the side chains of non-natural alpha amino acids whichcontain functional substituents, for example amino, carboxyl, hydroxy,mercapto, guanidyl, imidazolyl, or indolyl groups, such functionalsubstituents may optionally be protected.

The term “protected” when used in relation to a functional substituentin a side chain of a natural or non-natural alpha-amino acid means aderivative of such a substituent which is substantially non-functional.The widely used handbook by T. W. Greene and P. G. Wuts “ProtectiveGroups in Organic Synthesis” Second Edition, Wiley, New York, 1991reviews the subject. For example, carboxyl groups may be esterified (forexample as a C₁–C₆ alkyl ester), amino groups may be converted to amides(for example as a NHCOC₁–C₆ alkyl amide) or carbamates (for example asan NHC(═O)OC₁–C₆ alkyl or NHC(═O)OCH₂Ph carbamate), hydroxyl groups maybe converted to ethers (for example an OC₁–C₆ alkyl or a O(C₁–C₆alkyl)phenyl ether) or esters (for example a OC(═O)C₁–C₆ alkyl ester)and thiol groups may be converted to thioethers (for example atert-butyl or benzyl thioether) or thioesters (for example a SC(═O)C₁–C₆alkyl thioester).

There are several actual or potential chiral centres in the compoundsaccording to the invention because of the presence of asymmetric carbonatoms. The presence of several asymmetric carbon atoms gives rise to anumber of diastereoisomers with R or S stereochemistry at each chiralcentre. The invention includes all such diastereoisomers and mixturesthereof. Currently, the preferred stereoconfiguration of the carbon atomcarrying the R₂ group is R; that of the carbon atom carrying the R₄group (when asymmetric) is S; and that of the carbon atom carrying theR₁ group (when asymmetric) is R.

In the compounds of formula (I) as defined above for use according tothe invention, and in the novel compounds of the invention of formula(II) as defined above (but subject to the provisos therein):

-   R₁ may be, for example, hydrogen, methyl, or trifluoromethyl.    Hydrogen is currently preferred.-   R₂ may be, for example:    -   optionally substituted C₁–C₈ alkyl, C₃–C₆ alkenyl, C₃–C₆ alkynyl        or cycloalkyl;    -   phenyl(C₁–C₆ alkyl)-, phenyl(C₃–C₆ alkenyl)- or phenyl(C₃–C₆        alkynyl)-optionally substituted in the phenyl ring;    -   cycloalkyl(C₁–C₆ alkyl)-, cycloalkyl(C₃–C₆ alkenyl)- or        cycloalkyl(C₃–C₆ alkynyl)-optionally substituted in the        cycloalkyl ring;    -   heterocyclyl(C₁–C₆ alkyl)-, heterocyclyl(C₃–C₆ alkenyl)- or        heterocyclyl(C₃–C₆ alkynyl)- optionally substituted in the        heterocyclyl ring; or    -   CH₃(CH₂)_(p)O(CH₂)_(q)— or CH₃(CH₂)_(p)S(CH₂)_(q)—, wherein p is        0, 1, 2 or 3 and q is 1, 2 or 3.

Specific examples of R₂ groups include

-   -   methyl, ethyl, n- and iso-propyl, n- and iso-butyl, n-pentyl,        iso-pentyl 3-methyl-but-1-yl, n-hexyl, n-heptyl, n-acetyl,        n-octyl, methylsulfanylethyl, ethylsulfanylmethyl,        2-methoxyethyl, 2-ethoxyethyl, 2-ethoxymethyl, 3-hydroxypropyl,        allyl, 3-phenylprop-3-en-1-yl, prop-2-yn-1-yl,        3-phenylprop-2-yn-1-yl, 3-(2-chlorophenyl)prop-2-yn-1-yl,        but-2-yn-1-yl, cyclopentyl, cyclohexyl, cyclopentylmethyl,        cyclopentylethyl, cyclopentylpropyl, cyclohexylmethyl,        cyclohexylethyl, cyclohexylpropyl, furan-2-ylmethyl,        furan-3-methyl, tetrahydrofuran-2-ylmethyl,        tetrahydrofuran-2-ylmethyl, piperidinylmethyl, phenylpropyl,        4-chlorophenylpropyl, 4-methylphenylpropyl,        4-methoxyphenylpropyl, benzyl, 4-chlorobenzyl, 4-methylbenzyl,        and 4-methoxybenzyl.    -   Presently preferred groups at R₂ are n-propyl, n-butyl,        n-pentyl, benzyl and cyclopentylmethyl.

In the case of R₃, hydrogen is presently preferred.

-   R₄ may be, for example    -   the characterising group of a natural α amino acid, for example        benzyl, or 4-methoxyphenylmethyl, in which any functional group        may be protected, any amino group may be acylated and any        carboxyl group present may be amidated; or    -   a group -[Alk]_(n)R₉ where Alk is a (C₁–C₆)alkylene or        (C₂–C₆)alkenylene group optionally interrupted by one or more        —O—, or —S— atoms or —N(R₁₂)— groups [where R₁₂ is a hydrogen        atom or a (C₁–C₆)alkyl group], n is 0 or 1, and R₉ is hydrogen        or an optionally substituted phenyl, aryl, heterocyclyl,        cycloalkyl or cycloalkenyl group or (only when n is 1) R₉ may        additionally be hydroxy, mercapto, (C₁–C₆)alkylthio, amino,        halo, trifluoromethyl, nitro, —COOH, —CONH₂, —COOR^(A),        —NHCOR^(A), —CONHR^(A), —NHR^(A), NR^(A)R^(B), or CONR^(A)R^(B)        wherein R^(A) and R^(B) are independently a (C₁–C₆)alkyl group;        or    -   a benzyl group substituted in the phenyl ring by a group of        formula —OCH₂COR₈ where R₈ is hydroxyl, amino, (C₁–C₆)alkoxy,        phenyl(C₁–C₆)alkoxy, (C₁–C₆)alkylamino, di((C₁–C₆)alkyl)amino,        phenyl(C₁–C₆)alkylamino; or    -   a heterocyclic(C₁–C₆)alkyl group, either being unsubstituted or        mono- or di-substituted in the heterocyclic ring with halo,        nitro, carboxy, (C₁–C₆)alkoxy, cyano, (C₁–C₆)alkanoyl,        trifluoromethyl (C₁–C₆)alkyl, hydroxy, formyl, amino,        (C₁–C₆)alkylamino, di-(C₁–C₆)alkylamino, mercapto,        (C₁–C₆)alkylthio, hydroxy(C₁–C₆)alkyl, mercapto(C₁–C₆)alkyl or        (C₁–C₆)alkylphenylmethyl; or    -   a group —CR_(a)R_(b)R_(c) in which:    -   each of R_(a), R_(b) and R_(c) is independently hydrogen,        (C₁–C₆)alkyl, (C₂–C₆)alkenyl, (C₂–C₆)alkynyl,        phenyl(C₁–C₆)alkyl, (C₃–C₈)cycloalkyl; or    -   R_(c) is hydrogen and R_(a) and R^(b) are independently phenyl        or heteroaryl such as pyridyl; or    -   R_(c) is hydrogen, (C₁–C₆)alkyl, (C₂–C₆)alkenyl, (C₂–C₆)alkynyl,        phenyl(C₁–C₆)alkyl, or (C₃–C₈)cycloalkyl, and R_(a) and R_(b)        together with the carbon atom to which they are attached form a        3 to 8 membered cycloalkyl or a 5- to 6-membered heterocyclic        ring; or    -   R_(a), R_(b) and R_(c) together with the carbon atom to which        they are attached form a tricyclic ring (for example adamantyl);        or    -   R_(a) and R_(b) are each independently (C₁–C₆)alkyl,        (C₂–C₆)alkenyl, (C₂–C₆)alkynyl, phenyl(C₁-C₆)alkyl, or a group        as defined for R_(c) below other than hydrogen, or R_(a) and        R_(b) together with the carbon atom to which they are attached        form a cycloalkyl or heterocyclic ring, and R_(c) is hydrogen,        —OH, —SH, halogen, —CN, —CO₂H, (C₁–C₄)perfluoroalkyl, —CH₂OH,        —CO₂(C₁–C₆)alkyl, —O(C₁–C₆)alkyl, —O(C₂–C₆)alkenyl,        —S(C₁C₆)alkyl, —SO(C₁–C₆)alkyl, —SO₂(C₁–C₆) alkyl,        —S(C₂–C₆)alkenyl, —SO(C₂C₆)alkenyl, —SO₂(C₂–C₆)alkenyl or a        group —Q—W wherein Q represents a bond or —O—, —S—, —SO— or        —SO₂— and W represents a phenyl, phenylalkyl, (C₃–C₈)cycloalkyl,        (C₃–C₈)cycloalkylalkyl, (C₄–C₈)cycloalkenyl,        (C₄–C₈)cycloalkenylalkyl, heteroaryl or heteroarylalkyl group,        which group W may optionally be substituted by one or more        substituents independently selected from, hydroxyl, halogen,        —CN, —CO₂H, —CO₂(C₁–C₆)alkyl, —CONH₂, —CONH(C₁–C₆)alkyl,        —CONH(C₁–C₆alkyl)₂, —CHO, —CH₂OH, (C₁–C₄)perfluoroalkyl,        —O(C₁–C₆)alkyl, —S(C₁–C₆)alkyl, —SO(C₁–C₆)alkyl,        —SO₂(C₁–C₆)alkyl, —NO₂, —NH₂, —NH(C₁–C₆)alkyl,        —N((C₁–C₆)alkyl)₂, —NHCO(C₁–C₆)alkyl, (C₁–C₆)alkyl,        (C₂–C₆)alkenyl, (C₂–C₆)alkynyl, (C₃–C₈)cycloalkyl,        (C₄–C₈)cycloalkenyl, phenyl or benzyl.

Examples of particular R₄ groups include methyl, ethyl, benzyl,4-chlorobenzyl, 4-hydroxybenzyl, phenyl, cyclohexyl, cyclohexylmethyl,pyridin-3-ylmethyl, tert-butoxymethyl, naphthylmethyl, iso-butyl,sec-butyl, tert-butyl, 1-benzylthio-1-methylethyl,1-methylthio-1-methylethyl, 1-mercapto-1-methylethyl,1-methoxy-1-methylethyl, 1-hydroxy-1-methylethyl,1-fluoro-1-methylethyl, hydroxymethyl, 2-hydroxethyl, 2-carboxyethyl,2-methylcarbamoylethyl, 2-carbamoylethyl, and 4-aminobutyl. Presentlypreferred R₄ groups include tert-butyl, iso-butyl, benzyl and methyl.

R₃ and R₄ when taken together with the nitrogen and carbon atoms towhich they are respectively attached may form an optionally substitutedsaturated heterocyclic ring of 5 to 8 atoms. For example, R₃ and R₄ mayform a bridge between the nitrogen and carbon atoms to which they areattached, said bridge being represented by the divalent radical—(CH₂)₃₋₆—, or —(CH₂)_(r)—O—(CH₂)_(s)—, or —(CH₂)_(r)—S—(CH₂)_(s)—,wherein r and s are each independently 1, 2 or 3 with the proviso thatr+s=2, 3, 4, or 5.

R₅ and R₆ may independently be, for example, hydrogen, methyl, ethyl,tert-butyl, cyclopentyl, cyclohexyl, 1,1,3,3-tetramethylbutyl,benzyl, or2-hydroxyethyl; or R₅ and R₆ when taken together with the nitrogen atomto which they are attached may form a saturated 5- to 8-memberedmonocyclic N-heterocyclic ring which is attached via the N atom andwhich optionally contains —N(R₁₁)— wherein R₁₁ is hydrogen or C₁–C₆alkyl, benzyl, acyl, or an amino protecting group, O, S, SO or SO₂ as aring member, and/or is optionally substituted on one or more C atoms byhydroxy, C₁–C₆ alkyl, hydroxy(C₁–C₆ alkyl)-, C₁–C₆ alkoxy, oxo,ketalised oxo, amino, mono(C₁–C₆ alkyl)amino, di(C₁–C₆ alkyl)amino,carboxy, C₁–C₆ alkoxycarbonyl, hydroxymethyl, C₁–C₆ alkoxymethyl,carbamoyl, mono(C₁–C₆ alkyl)carbamoyl, di(C₁–C₆ alkyl)carbamoyl, orhydroxyimino.

Examples of such rings are substituted or unsubstituted 1-pyrrolidinyl,piperidin-1-yl, 1-piperazinyl, hexahydro-1-pyridazinyl, morpholin-4-yl,tetrahydro-1,4-thiazin-4-yl, tetrahydro-1,4-thiazin-4-yl 1-oxide,tetrahydro-1,4-thiazin-4-yl 1,1-dioxide, hexahydroazipino, oroctahydroazocino. Substituted examples of the foregoing are2-(methylcarbamoyl)-1-pyrrolidinyl, 2-(hydroxymethyl)-1-pyrrolidinyl,4-hydroxypiperidino, 2-(methylcarbamoyl)piperidino,4-hydroxyiminopiperidino, 4-methoxypiperidino, 4-methylpiperidin-1yl,4-benzylpiperidin-1-yl, 4-acetylpiperidin-1-yl,4-methyl-1-piperazinyl,4-phenyl-1-piperazinyl, 1,4-dioxa-8-azaspiro[4,5]decan-8-yl,hexahydro-3-(methylcarbamoyl)-2-pyridazinyl, andhexahydro-1-(benzyloxycarbonyl)-2-pyridazinyl,decahydroisoquinolin-2-yl, and 1,2,3,4-tetrahydroisoquinolin-2-yl.

When A is a group of formula (IA), it is currently preferred that R₅ bemethyl or hydrogen, and R₆ be methyl.

R₇ may be, for example, hydrogen, or a group R₂₀C(O)— where R₂₀ is a(C₁–C₆)alkyl group such as methyl or ethyl.

Specific examples of compounds useful as antibacterial agents inaccordance with the invention include those of the specific Examplesherein. Preferred novel compounds of the invention include

-   -   2R (orS)-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid        (1S-dimethylcarbamoyl-ethyl)-amide and    -   2R (or        S)-[(Formyl-hydroxy-amino)-methyl]-3-cyclopentyl-propionic acid        (1S-dimethyl-carbamoyl-2,2-dimethyl-propyl)-amide        and their pharmaceutically and veterinarily acceptable salts.

Compounds with which the invention is concerned the invention may beprepared by deprotecting an O-protected N-formyl-N-hydroxyamino compoundof formula (II):

in which R₁, R₂, and A are as defined in general formula (I) and R₂₅ isa hydroxy protecting group removable to leave a hydroxy group byhydrogenolysis or hydrolysis. Benzyl is a preferred R₂₅ group forremoval by hydrogenolysis, and tert-butyl and tetrahydropyranyl arepreferred groups for removal by acid hydrolysis.

Compounds of formula (II) wherein A is a group of formula (IA), (IB),(IC) or (ID) may be prepared by causing an acid of formula (III) or anactivated derivative thereof to react with an amine of formula (IVA),(IVB), (IVC) or (IVD) respectively

wherein R₁ R₂, R₃, R₄, R₅, R₆ and R₇ are as defined in general formula(I) except that the —OH group in (IVB) and any substituents in R₁ R₂,R₃, R₄, R₅, R₆ and R₇ which are potentially reactive in the couplingreaction may themselves be protected from such reaction, and R₂₅ is asdefined in relation to formula (II) above, and optionally removingprotecting groups from the —OH group in (IVB) and R₁ R₂, R₃, R₄, R₅, R₆and R₇.

Compounds of formula (III) may be prepared by N-formylation, for exampleusing acetic anhydride and formic acid, or 1-formylbenzotriazole, ofcompounds of formula (V)

wherein R₁, R₂ and R₂₅ are as defined in relation to formula (II) and Xis either a chiral auxiliary or an OR₂₆ group wherein R₂₆ is hydrogen ora hydroxy protecting group. In the case where X is an OR₂₆ group or achiral auxiliary the hydroxy protecting group or auxiliary is removedafter the formylation step to provide the compound of formula (V).Suitable chiral auxiliaries include substituted oxazolidinones which maybe removed by hydrolysis in the presence of base.

In an alternative procedure compounds of general formula (II) may beprepared by N-formylation, for example using acetic anhydride and formicacid, or 1-formylbenzotriazole, of compounds of formula (VI)

wherein R₁, R₂, R₂₅ and A are as defined in relation to formula (II).

Compounds of formula (VI) wherein A is a group of formula (IA), (IB),(IC) or (ID) may be prepared by causing an acid of general formula (VII)or an activated derivative thereof

wherein R₁, R₂ and R₂₅ are as defined in relation to formula (II) toreact with an amine of formula (IVA), (IVB), (IVC) or (IVD) respectivelyas defined above.

Alternatively compounds of general formula (VI) may be prepared byreduction of an oxime of general formula (VIII).

Reducing agents include certain metal hydrides (e.g. sodiumcyanoborohydride in acetic acid, triethylsilane or borane/pyridine) andhydrogen in the presence of a suitable catalyst.

In an alternative procedure compounds of general formula (II) wherein R₁and R₂ are as defined in general formula (I), R₂₅ is a hydroxyprotecting group as defined above and A is a group of formula (IA)wherein R₃, R₄, R₅ are as defined in general formula (IA) and R₆ ishydrogen may be prepared by a 4-component Ugi reaction of carboxylicacid of general formula (III) as defined above, an amine of formula(IX), an aldehyde of formula (X) and an isonitrile of formula (XI)R₃—NH₂  (IX)R₄—CHO  (X)R₅—CN  (XI)wherein R₃, R₄ and R₅ are as defined above.

A compound of general formula (V) may be prepared by reduction of anoxime of general formula (XI)

wherein R₁, R₂, and R₂₅ are as defined above, and X is either an OR₂₆group as defined above or a chiral auxiliary. Reducing agents includecertain metal hydrides (eg sodium cyanoborohydride in acetic acid,triethylsilane or borane/pyridine) and hydrogen in the presence of asuitable catalyst. Following the reduction when the group X is a chiralauxiliary it may be optionally converted to a OR₂₆ group.

A compound of general formula (XI) can be prepared by reaction of aβ-keto carbonyl compound of general formula (XII)

wherein R₁, R₂, and X are as defined above, with an O-protectedhydroxylamine.

β-keto carbonyl compounds (XII) may be prepared in racemic form byformylation or acylation of a carbonyl compound of general formula(XIII)

wherein R₂ and X are as defined above, with a compound of generalformula (XIV)

wherein R₁ is as defined above and Z is a leaving group such as halogenor alkoxy, in the presence of a base.

Another method for the preparation of a compound of general formula (V)is by Michael addition of a hydroxylamine derivative to α,β-unsaturatedcarbonyl compounds of general formula (XV)

wherein R₁, R₂, and X are as defined above. Following the Michaeladdition reaction, when the group X is a chiral auxiliary it may beoptionally converted to a OR₂₆ group. The α,β-unsaturated carbonylcompounds (XV) may be prepared by standard methods.

Salts of the compounds of the invention include physiologicallyacceptable acid addition salts for example hydrochlorides,hydrobromides, sulphates, methane sulphonates, p-toluenesulphonates,phosphates, acetates, citrates, succinates, lactates, tartrates,fumarates and maleates. Salts may also be formed with bases, for examplesodium, potassium, magnesium, and calcium salts.

Compositions with which the invention is concerned may be prepared foradministration by any route consistent with the pharmacokineticproperties of the active ingredient(s).

Orally administrable compositions may be in the form of tablets,capsules, powders, granules, lozenges, liquid or gel preparations, suchas oral, topical, or sterile parenteral solutions or suspensions.Tablets and capsules for oral administration may be in unit dosepresentation form, and may contain conventional excipients such asbinding agents, for example syrup, acacia, gelatin, sorbitol,tragacanth, or polyvinyl-pyrrolidone; fillers for example lactose,sugar, maize-starch, calcium phosphate, sorbitol or glycine; tablettinglubricant, for example magnesium stearate, talc, polyethylene glycol orsilica; disintegrants for example potato starch, or acceptable wettingagents such as sodium lauryl sulphate. The tablets may be coatedaccording to methods well known in normal pharmaceutical practice. Oralliquid preparations may be in the form of, for example, aqueous or oilysuspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for reconstitution with water or othersuitable vehicle before use. Such liquid preparations may containconventional additives such as suspending agents, for example sorbitol,syrup, methyl cellulose, glucose syrup, gelatin hydrogenated ediblefats; emulsifying agents, for example lecithin, sorbitan monooleate, oracacia; non-aqueous vehicles (which may include edible oils), forexample almond oil, fractionated coconut oil, oily esters such asglycerine, propylene glycol, or ethyl alcohol; preservatives, forexample methyl or propyl p-hydroxybenzoate or sorbic acid, and ifdesired conventional flavouring or colouring agents.

For topical application to the skin, the active ingredient(s) may bemade up into a cream, lotion or ointment. Cream or ointment formulationswhich may be used for the drug are conventional formulations well knownin the art, for example as described in standard textbooks ofpharmaceutics such as the British Pharmacopoeia.

The active ingredient(s) may also be administered parenterally in asterile medium. Depending on the vehicle and concentration used, thedrug can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as a local anaesthetic, preservative andbuffering agents can be dissolved in the vehicle. Intra-venous infusionis another route of administration for the compounds used in accordancewith the invention.

Safe and effective dosages for different classes of patient and fordifferent disease states will be determined by clinical trial as isrequired in the art. It will be understood that the specific dose levelfor any particular patient will depend upon a variety of factorsincluding the activity of the specific compound employed, the age, bodyweight, general health, sex, diet, time of administration, route ofadministration, rate of excretion, drug combination and the severity ofthe particular disease undergoing therapy.

The finding that compounds with PDF inhibitory activity can inhibit orprevent bacterial growth, opens up a novel approach for identifying newantibacterial agents by screening test compounds for activity asinhibitors of PDF in vitro, followed by confirmation of theirantibacterial ability using bacterial growth inhibition studies. Thisfinding also makes available (i) the use of compounds with PDFinhibitory activity as antibacterial agents, and (ii) a method for thetreatment of bacterial infection or contamination by applying oradministering a compound which inhibits the activity of bacterial PDF.

According to a further aspect of the invention therefore, there isprovided a method for the identification of antibacterial compounds,comprising screening test compounds for their ability to inhibit PDF invitro, selecting those compounds which exhibit said ability and testingthese for their ability to inhibit bacterial growth. The ability toinhibit bacterial growth can be performed using classical plate or brothculture bacterial growth inhibition studies, such as those performed inthe Biological Examples herein.

A suitable in vitro PDF inhibition screen may comprise mixing togetherPDF, a PDF substrate, preferably, labelled with a detectable marker, andthe test compound and assessing after a suitable length of time whetheror not the presence of the test compound inhibits the ability of PDF todeformylate the substrate.

In a preferred embodiment, the cleaved substrate is detected with afluorogenic marker such as fluorescamine. On removal of the formyl groupfrom the N-terminal methionine of the PDF substrate, the free aminogroup reacts with fluorescamine generating a fluorescent product.

An alternative screen involves assessing whether a protein expressed bybacteria that express endogenous (or recombinantly expressed) PDF, whengrown in the presence of a test compound, yields suitable substrate forN-terminal sequencing, or yields a lesser amount of substrate, thanprotein expressed from the same bacteria grown in the absence of thetest compound. Such a method could be based on that used in theBiological Examples herein.

The person skilled in the art will be able to develop, without inventiveinput, alternative methods for screening test compounds for theirability to inhibit bacterial PDF.

The natural antibiotic actinonin (see for example J. C. S Perkin I,1975, 819) is a hydroxamic acid derivative of Structure (A):

In addition to actinonin, various structural analogues of actinonin havealso been shown to have antibacterial activity (see for exampleBroughton et al. (Devlin et al. Journal of the Chemical Society. PerkinTransactions 1 (9):830–841, 1975; Broughton et al. Journal of theChemical Society. Perkin Transactions 1 (9):857–860, 1975)

To date, however, the mechanism underlying the antibacterial activity ofactinonin has not been known. The present inventors have found thatactinonin inhibits the activity of bacterial PDF.

The matlystatin group of compounds share a number of structuralsimilarities with actinonin. Both are peptidic molecules with functionalhydroxamic acid metal binding groups (Ogita et al., J. Antibiotics.45(11):1723–1732; Tanzawa et al., J. Antibiotics. 45(11):1733–1737;Haruyama et al., J. Antibiotics. 47(12):1473–1480; Tamaki et al., J.Antibiotics. 47(12):1481–1492). The matlystatins and their closestructural analogues are characterised by the presence in the moleculeof a divalent piperazin-1,6-diyl group, i.e.

In view of their close structural similarity to actinonin, theobservation that actinonin inhibits PDF implies that matlystatincompounds may also inhibit PDF.

According to a further aspect of the present invention there is providedthe use of a compound which inhibits the activity of bacterial PDF, inthe preparation of an antibacterial composition or agent, provided that(i) the compound is not of formula (XI)RCO—CH(W)—NH—CO—CH(Y)—CH₂—CO—NH—OH  (XI)wherein,

-   (a) R is a cyclic amino group, W is hydrogen, methyl, isopropyl,    isobutyl or benzyl, and Y is hydrogen, C₁-C₆ alkyl, phenyl, benzyl,    4-chlorophenylmethyl, 4-nitrophenylmethyl, or 4-aminophenylmethyl;    or,-   (b) R is 2-pyridylamino or 2-thiazolylamino, W is isopropyl and Y is    n-pentyl; or,-   (c) R is diethylamino, W is methyl or isopropyl and Y is n-pentyl;    or (ii) the compound is not one containing a divalent    piperazin-1,6-diyl group, i.e. a group of formula (XII):

According to a further aspect of the invention there is provided amethod of treating bacterial infection or contamination by administeringto a patient suffering such infection or contamination, or applying tothe site of such infection or contamination, an antibacteriallyeffective amount of a compound which inhibits the activity of bacterialPDF enzyme, provided that the compound is not one provided in theprovisos in the immediately preceeding paragraph.

These provisos exclude actinonin and its antibacterially activeanalogues as disclosed in Devlin et al., Journal of the ChemicalSociety. Perkin Transactions 1 (9):830–841, 1975 and Broughton et al.Journal of the Chemical Society. Perkin Transactions 1 (9):857–860,1975, and the matlystatin compounds disclosed in Ogita et al., J.Antibiotics. 45(11):1723–1732; Tanzawa et al., J. Antibiotics.45(11):1733–1737; Haruyama et al., J. Antibiotics. 47(12):1473–1480 andTamaki et al., J. Antibiotics. 47(12):1481–1492.

The following examples illustrate embodiments of the invention.L-tert-Leucine-N-methylamide and L-tert-leucine-N,N-dimethylamide andother amino acid derivatives were prepared according to establishedliterature methods.

The following abbreviations have been used throughout:

-   DMF N,N-Dimethylformamide-   EDC N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride-   HOAt 1-Hydroxy-7-aza-benzotriazole-   HOBt 1-Hydroxybenzotriazole-   HPLC High performance liquid chromatography-   LRMS Low resolution mass spectrometry-   TLC Thin layer chromatography

¹H and ¹³C NMR spectra were recorded using a Bruker AC 250E spectrometerat 250.1 and 62.9 MHz, respectively. Mass spectra were obtained using aPerkin Elmer Sciex API 165 spectrometer using both positive and negativeion modes. Infra-red spectra were recorded on a Perkin Elmer PE 1600FTIR spectrometer.

EXAMPLE 1

2R (or S)-[(Formyl-hydroxy-amino)-methyl]-hexanoicacid-(2,2-dimethyl-1S-methyl-carbamoyl-propyl)-amide

The title compound was prepared according to the route outlined inScheme 1 and as described in detail below:

STEP A: 2-Butyl-acrylic acid

Butylmalonic acid (25 g, 156 mmol) was dissolved in ethanol (250 ml) and37% formaldehyde solution (15.45 ml, 156 mmol) was added followed bypiperidine (47 ml, 624 mmol). The mixture was stirred overnight at 80°C. under a reflux condenser. The solvents were removed under reducedpressure and the residue was diluted with 1M hydrochloric acid andextracted with dichloromethane (3×30 ml). The combined organic extractswere washed with brine, dried over anhydrous magnesium sulfate, filteredand evaporated to afford the desired product as a yellow oil (25 g, withresidual solvent). ¹H-NMR: δ (CDCl₃), 10.04 (1H, br s), 6.22 (1H, s),5.57 (1H, d, J=1.3 Hz), 2.30 (2H, t, J=6.9 Hz), 1.38 (4H, m), and 0.91(3H, t, J=7.2 Hz).

STEP B: 2RS-(Benzyloxy-amino-methyl)-hexanoic acid

A mixture of 2-butyl-acrylic acid (3.43 g, 27.1 mmol) andO-benzylhydroxylamine (5 g, 40.65 mmol) were heated at 80° C. overnight.The mixture was cooled to room temperature, diluted with ethyl acetate(40 ml), and washed with 1M hydrochloric acid (3×20 ml), saturatedsodium hydrogen carbonate solution (2×20 ml) and brine (2×20 ml), driedover anhydrous magnesium sulfate, filtered and evaporated to leave thetitle compound as a white solid (2.62 g, 39%). ¹H-NMR: δ (CDCl₃), 8.05(1H, br s), 7.35 (5H, m), 5.00 (2H, m), 3.28 (2H, m), 2.98 1H, m), 1.31(6H, m) and 0.88 (3H, t, J=5.0 Hz).

Step C: 2RS-[(Benzyloxy-formyl-amino)-methyl]-hexanoic acid

2RS-(Benzyloxyamino-methyl)-hexanoic acid (2.62 g, 10.51 mmol) wasdissolved in formic acid (4 ml, 105 mmol) and acetic anhydride (1.9 ml,21.02 mmol) and stirred overnight at room temperature. The solution wasdiluted with ethyl acetate (40 ml), washed with water (2×20 ml),saturated sodium hydrogen carbonate solution (20 ml) and brine (20 ml),dried over anhydrous magnesium sulfate, filtered and evaporate to leavethe desired product as a yellow oil (2.9 g, 99%). ¹H-NMR: δ (CDCl₃,rotamers), 8.21 (0.5H, s), 8.14 (0.5H, s), 7.37 (5H, m), 4.98 (2H, m),3.86 (1H, m), 3.27 (0.5H, dd, J=6.0, 14.0 Hz), 2.93 (0.5H, m), 2.77 (1H,m), 1.50 (2H, m), 1.30 (4H, m) and 0.88 (3H, m).

STEP D: 2RS-[(Benzyloxy-formyl-amino)-methyl]-hexanoic acidpentafluorophenyl ester

2RS-[(Benzyloxy-formyl-amino)-methyl]-hexanoic acid (30.72 g, 110 mmol)and penta-fluorophenol (26.31 g, 143 mmol) were dissolved indichloromethane (150 ml) and the solution was stirred and cooled in anice bath during addition of EDC (25.3 g, 131 mmol). The reaction mixturewas allowed to warm to room temperature and stirred overnight. Thesolution was washed successively with 1M hydrochloric acid (2×50 ml),0.5M sodium carbonate (2×50 ml) and brine (50 ml), dried over anhydrousmagnesium sulfate and filtered. The filtrate was evaporated underreduced pressure and the residue was purified by flash chromatography(silica gel, dichloromethane) to afford the title compound as acolourless oil (15.0 g, 31%). ¹H-NMR: δ (CDCl₃, rotamers), 8.17 (1H, brs), 7.37 (5H, m), 4.95–4.70 (2H, br m), 4.10–3.75 (2H, br m), 3.10 (1H,br s), 1.88–1.55 (2H, m), 1.39 (4H, m) and 0.92 (3H, t, J=7.0 Hz).

STEP E: 2R (or S)-[(Benzyloxy-formyl-amino)-methyl]-hexanoicacid-(2,2-dimethyl-1-methyl-carbamoyl-propyl)-amide

2RS-[(Benzyloxy-formyl-amino)-methyl]-hexanoic acid pentafluorophenylester (5 g, 11 mmol) and tert-leucine N-methylamide (1.62 g, 11 mmol)were dissolved in DMF (60 ml) and the mixture was stirred overnight at35 ° C. The solvent was removed under reduced pressure and the residuewas redissolved in dichloromethane. The solution was washed successivelywith 0.5 M sodium carbonate, 1.0 M hydrochloric acid and brine, driedover anhydrous magnesium sulfate and filtered. The two diastereoisomericproducts were separated by flash chromatography (silica gel, gradientelution with 30% to 0% hexane in ethyl acetate). Diastereoisomer A(higher R_(f)): ¹H-NMR: δ (CDCl₃, rotamers), 8.12, 7.87 (1H, 2br s),7.27 (5H, m), 6.26 (1H, d, J=8.7 Hz), 5.78 (1H, br s), 4.91–4.60 (2H, brm), 4.15 (1H, d, J=9.2 Hz), 3.75 (2H, br m), 2.79 (3H, d, J=4.8 Hz),2.56 (1H, m), 1.60–1.35 (2H, br m), 1.24 (4H, m), 0.96 (9H, s) and 0.86(3H, t, J=6.7 Hz). Diastereoisomer B (lower R_(f)): ¹H-NMR: δ (CDCl₃,rotamers), 8.16, 7.88 (1H, 2br s), 7.27 (5H, m), 6.28 (1H, d, J=8.9 Hz),5.70–5.44 (1H, br s), 4.98–4.61 (2H, br m), 4.14 (1H, d, J=9.2 Hz),3.78–3.62 (2H, br m), 2.85–2.60 (3H, br m), 2.47 (1H, m), 1.72–1.25 (6H,br m), 0.98 (9H, s) and 0.88 (3H, t, J=6.7 Hz).

STEP F: 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-hexanoicacid-(2,2-dimethyl-1S-methylcarbamoyl-propyl)-amide

2-[(Benzyloxy-formyl-amino)-methyl]-hexanoicacid-(2,2-dimethyl-1-methylcarbamoyl-propyl)-amide (diastereoisomer A)(1.0 g, 2.5 mmol) was dissolved in a mixture of ethyl acetate (20 ml)and ethanol (1 ml) and the solution was placed under an argonatmosphere. 10% palladium on charcoal was added and a fine stream ofhydrogen gas was bubbled through the suspension. After 40 minutes TLCanalysis revealed that all the starting material had been consumedleaving a more polar, ferric chloride positive species. The system wasflushed with argon before removing the catalyst by filtration. Thefiltrate was evaporated to dryness to leave the title compound as anoff-white foam (810 mg, including residual solvent). ¹H-NMR: δ((CD₃)₂SO, rotamers), 9.81, 9.41 (1H, 2br s), 7.82–7.60 (3H, m), 4.04(1H, d, J=9.3 Hz), 3.50–3.02 (2H, m), 2.87–2.60 (1H, m), 2.41 (3H, d,J=4.5 Hz), 1.39–0.93 6H, m), 0.75 (9H, s) and 0.67 (3H, t, J=5.7 Hz).¹³C-NMR: δ ((CD₃)₂SO), 172.5, 170.2, 157.5, 59.9, 42.8, 33.3, 29.0,28.4, 28.2, 26.4, 24.8, 21.7 and 13.3. IR (KBr disc), v_(max) 3309,2959, 2873, 1646 and 1540 cm⁻¹.

2-[(Benzyloxy-formyl-amino)-methyl]-hexanoicacid-(2,2-dimethyl-1-methylcarbamoyl-propyl)-amide (diastereoisomer B)(1.0 g, 2.5 mmol) was similarly deprotected to give diastereoisomer B ofthe title compound (740 mg, 97%). ¹H-NMR: δ ((CD₃)₂SO, rotamers), 9.75,9.30 (1H, 2br s), 7.81–7.42 (3H, m), 4.04 (1H, d, J=9.5 Hz), 3.53–3.02(2H, m), 2.80–2.55 (1H, m), 2.41 (3H, d, J=4.5 Hz), 1.33–0.82 (6H, m),0.72 (9H, s) and 0.67 (3H, t, J=6.7 Hz). ¹³C-NMR: δ ((CD₃)₂SO), 172.6,170.4, 161.7, 157.0, 59.8, 34.0, 29.4, 28.6, 26.7, 25.2, 22.1 and 14.1.IR (KBr disc), v_(max) 3312, 2959, 1640, 1541, 1369 and 1240 cm⁻¹.

EXAMPLE 2 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-hexanoicacid-(2,2-dimethyl-1S-tert-butyl-carbamoyl-propyl)-amide

The title compounds were prepared by analogy with Example 1, using theL-tert-leucine-N-tert-butylamide in place ofL-tert-leucine-N-methylamide in Step E. The diastereoisomers were notseparable by flash chromatography (silica gel, ethyl acetate) at Step Eand were converted to a mixture of the desired N-formyl hydroxylaminederivatives by hydrogenolysis. White solid. ¹³C-NMR: δ ((CD₃)₂SO),172.8, 172.5, 170.1, 169.6, 161.6, 156.9, 59.9, 59.7, 51.9, 51.7, 50.2,49.6, 48.3, 43.2, 43.1, 42.7, 34.2, 34.0, 29.6, 29.3, 29.2, 28.8, 28.6,26.7, 22.2, 22.1, 20.3 and 13.9. IR (KBr), v_(max) 3311, 2964, 1639,1548, 1456, 1394, 1364 and 1226 cm⁻¹.

EXAMPLE 3 2R (orS)-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-methyl-2-morpholin-4-yl-2-oxo-ethyl)-amide

A solution of 2RS-[(benzyloxy-formyl-amino)-methyl]-hexanoic acidpentafluoro-phenyl ester (Example 1, Step D) (445 mg, 1 mmol) in DMF (5ml) was added to L alanine-N-morpholinoamide (158 mg, 1 mmol) in aboiling tube and stirred at 35° C. overnight. DMF was removed in vacuoand the residue was redissolved in dichloromethane (2 ml) and passedthrough a purification cartridge (Isolute-NH₂), eluting withdichloromethane (4 ml) in order to remove pentafluorophenol.Dichloromethane was removed under reduced pressure and the residue wasredissolved in formic acid (2 ml) and ethyl acetate (2 ml). The solutionwas then treated with 10% palladium on charcoal (200 mg) and stirred atroom temperature for 2 hours. Catalyst was removed by filtration throughcelite, washing well with methanol and solvents were removed in vacuo.Compounds were purified by reverse phase HPLC (gradient elution, 10–90%acetonitrile/water). Diastereoisomer A: ¹H-NMR; δ (CD₃OD), 8.03 (0.5H,s), 7.84 (0.5H, s), 4.75 (1H, m), 3.65 (8H, m), 3.39 (1H, m), 3.24 (1H,dd, J=4.0, 13.2 Hz), 2.84 (1H, m), 1.57 (2H, m), 1.34 (7H, m), and 0.92(3H, m). LRMS: −ve ion 328 [M−H]. Diastereoisomer B: ¹H-NMR; δ (CD₃OD),3.66 (8H, m), 3.41 (1H, dd, J=9.98, 13.1 Hz), 3.23 (1H, m), 2.90 (0.5H,m), 2.71 (0.5H, m), 1.62 (2H, m), 1.33 (7H, m), and 0.92 (3H, t, J=6.7Hz). LRMS: −ve ion 328 [M−H].

The compounds of Examples 4 to 12 were prepared by analogy with Example3 using the appropriate amine component in place ofL-alanine-N-morpholinoamide. Where both diastereoisomers were prepared,diastereoisomer A is the faster eluting and more potent against PDF invitro. In some cases only the faster running diastereoisomer was takenthrough to the final product.

EXAMPLE 4 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-dimethylcarbamoyl-ethyl)-amide

Diastereoisomer A: ¹H-NMR; δ (CD₃OD), 4.72 (1H, m), 3.53 (1H, dd, J=8.9,13.0 Hz), 3.23 (1H, m), 3.14 (3H, s), 2.95 (3H, s), 2.83 (0.5H, m), 2.74(0.5H, m), 1.57 (2H, m), 1.33 (7H, m) and 0.92 (3H, m). LRMS; +ve ion288 [M+H], −ve ion 286 [M−H].

Diastereoisomer B: ¹H-NMR; δ (CD₃OD), 4.74 (1H, m), 3.41 (1H, dd, J=9.9,13.0 Hz), 3.25 (1H, dd, J=4.0, 13.1 Hz), 3.15 (3H, s), 2.97 (3H, s),2.89 (0.5H, m), 2.72 (0.5H, m), 1.53 (2H, m), 1.33 (7H, m) and 0.93 (3H,t, J=6.7 Hz). LRMS: +ve ion 310 [M+Na], −ve ion 286 [M−H].

EXAMPLE 5 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-hydroxymethyl-3-methyl-butyl)-amide

Diastereoisomer A: ¹H-NMR; δ (CD₃OD), 4.07 (1H, m), 3.55 (1H, m), 3.45(2H, m), 3.20 (1H, m), 2.85 (0.5H, m), 2.80 (0.5H, m), 1.60 (3H, m),1.35 (6H, m) and 0.93 (9H, m). LRMS: +ve ion 289 [M+H], −ve ion 287[M−H].

Diastereoisomer B: ¹H-NMR; δ (CD₃OD), 4.07 (1H, m), 3.59 (1H, m), 3.45(2H, m), 3.24 (1H, m), 2.70 (1H, m), 1.62 (3H, m), 1.35 (6H, m) and 0.93(9H, m). LRMS: +ve ion 311 [M+Na], 289 [M+H], −ve ion 287 [M−H].

EXAMPLE 6 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-hydroxymethyl-2-phenyl-ethyl)-amide

Diastereoisomer A: ¹H-NMR; δ (CD₃OD), 7.24 (5H, m), 4.15 (1H, m), 3.54(2H, d, J=5.4 Hz), 3.38 (1H, dd, J=7.8, 13.1 Hz), 3.14 (1H, dd, J=4.7,13.2 Hz), 2.95 (1H, dd, J=7.3, 13.7 Hz), 2.68 (2H, m), 1.58 (2H, m),1.32 (4H, m), and 0.91 (3H, t, J=6.7 Hz). LRMS: +ve ion 345 [M+Na], 323[M+H], −ve ion 321 [M−H].

Diastereoisomer B: LRMS: +ve ion 345 [M+Na], 323 [M+H], −ve ion 321[M−H].

EXAMPLE ∂ 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid[2,2-dimethyl-1S-pyridin-2-yl-carbamoyl)-propyl]-amide

Diastereoisomer A: colourless oil. ¹H-NMR; δ (CD₃OD), 8.34 (1H, m), 8.06(1H, m), 7.90 (1H, m), 7.33 (1H, m), 4.45 (1H, s), 3.55 (1H, dd, J=8.3,13.2 Hz), 3.25 (1H, m), 3.05 (1H, m), 1.61 (2H, m), 1.32 (4H, m), 1.11(9H, s) and 0.85 (3H, m). LRMS: +ve ion 379 [M+H], −ve ion 377 [M−H].

Diastereoisomer B: colourless oil. ¹H-NMR; δ (CD₃OD), 8.33 (1H, m), 8.20(0.5H, m), 7.93 (1H, m), 7.41 (0.5H, m), 7.28 (1H, m), 4.48 (1H, s),3.52 (1H, dd, J=8.8, 13.1 Hz), 3.23 (1H, dd, J=3.9, 13.1 Hz), 3.05(0.5H, m), 2.87 (0.5H, m), 1.62 (2H, m), 1.36 (4H, m), 1.11 (9H, s) and0.93 (3H, m). LRMS: +ve ion 393 [M+Na], 379 [M+H], −ve ion 377 [M−H].

EXALMPLE 8 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-dimethylcarbamoyl-2-methyl-propyl) amide

Diastereoisomer A: colourless oil. LRMS: +ve ion 338 [M+Na], −ve ion 319[M−H].

EXAMPLE 9 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-dimethylcarbamoyl-2-phenyl-ethyl) amide

Diastereoisomer A: colourless oil. LRMS: +ve ion 386 [M+Na], −ve ion 362[M−H]

Diastereoisomer B: colourless oil. LRMS: +ve ion 386 [M+Na], −ve ion 362[M−H]

EXAMPLE 10 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-dimethylcarbamoyl-3-methyl-butyl) amide

Diastereoisomer A: colourless oil. LRMS: +ve ion 352 [M+Na], −ve ion 328[M−H].

EXAMPLE 11 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid[3-methyl-1S-pyrrolidine-1-carbonyl)-butyl] amide

Diastereoisomer A: colourless oil. LRMS: −ve ion 354 [M−H].

EXAMPLE 12 1-{2R (orS)-[(Formyl-amino)-methyl]-hexanoyl}-pyrrolidine-2S-carboxylic aciddimedthylamide

Diastereoisomer A: colourless oil. LRMS: +ve ion 336 [M+Na], −ve ion 312[M−H].

Disatereoisomer B: colourless oil. LRMS: +ve ion 336 [M+Na], −ve ion 312[M−H].

EXAMPLE 13 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-hexanoicacid-(1S-dimethylcarbamoyl-2,2-dimethyl-propyl)-amide

Method I

A synthetic route to the title compound is outlined in Scheme 2 and isdescribed in detail below.

Step A: 2RS-Formyl-heptanoic acid ethyl ester

Sodium metal (4.38 g, 0.191 mmol) was cut into small pieces and placedin a two-neck oven-dried round bottom flask under a blanket of argon.Anhydrous diethyl ether (100 ml) was added and the suspension wasstirred and cooled to 0° C. The flask was fitted with a reflux condenserbefore dropwise addition of ethanol (1.03 ml, 17.3 mmol). A mixture ofethyl formate (15.41 g, 0.208 mmol) and ethyl caproate (25 g, 0.173mmol) was added dropwise via a dropping funnel over a period of about 20minutes. The resulting orange suspension (sodium metal still visible)was allowed to warm to room temperature and stirred overnight. Theresulting thick orange suspension (no sodium metal visible) was cooledto 0° C. and diluted with ice-cold water (100 ml). The mixture wastransferred to a separating funnel and the aqueous phase was removed,washed with diethyl ether, cooled to 0° C. and acidified with 1 Mhydrochloric acid (200 ml). The emulsion was extracted with ethylacetate and the organic layer was separated, washed with brine, driedover anhydrous magnesium sulfate and filtered. The filtrate wasconcentrated under reduced pressure to give a yellow oil containingprimarily the title compound (11.09 g), which was used without furtherpurification in Step B.

Step B: 2RS-(Benzyloxyimino-methyl)-heptanoic acid ethyl ester

The crude Claisen product from Step A (11.0 g, 63.9 mmol) was dissolvedin ethanol (100 ml) and water (10 ml) and cooled to 0° C. during theaddition of sodium acetate (6.2 g, 76.6 mmol) and O-benzyl hydroxylaminehydrochloride (12.23 g, 76.6 mmol). The mixture was allowed to warm toroom temperature and stirred overnight. The resulting suspension wasfiltered and the filtrate was concentrated under reduced pressure. Theresidual yellow paste was partitioned between ethyl acetate and water.The organic layer was washed with 1 M hydrochloric acid and brine, driedover anhydrous magnesium sulfate, filtered and evaporated to a yellowoil. The desired product was obtained by flash chromatography (silicagel, gradient elution with 10% to 25% ethyl acetate in hexane. Yield9.19 g (52%). ¹H-NMR: δ (CDCl₃, mixture of syn- and anti-isomers), 7.46(0.6H, d, J=8.0 Hz), 7.38–7.28 (5H, m), 6.79 (0.4H, d, J=7.1 Hz), 5.11(0.8H, s), 5.08 (1.2H, s), 4.16 (1.2H, q, J=7.0 Hz), 4.13 (0.6H, q,J=7.0 Hz), 3.91 (0.4H, q, J=7.2 Hz), 3.21 (0.6H, td, J=8.0 and 6.1 Hz),1.90–1.48 (2H, m), 1.37–1.20 (7H, m), 0.87 (3H, t, J=7.0 Hz).

Step C: 2RS-(Benzyloxyimino-methyl)-heptanoic acid

2RS-(Benzyloxyimino-methyl)-heptanoic acid ethyl ester (7.0 g, 25.24mmol) was dissolved in methanol (125 ml) and the solution was cooled to0° C. 1 M Sodium hydroxide (26 ml, 26 mmol) was added in portions over 2minutes to give a pale yellow emulsion. Additional methanol was addeduntil a clear solution was obtained. The solution was allowed to stirfor 90 minutes at 0° C. then for 5 hours at room temperature whereuponTLC analysis suggested that all of the starting material had beenconsumed. The solvent was removed under reduced pressure and the residuewas partitioned between water and ethyl acetate. The aqueous layer wascooled to 0° C. and acidified with 1 M hydrochloric acid. The emulsionthus formed was extracted twice with ethyl acetate. The combined organicextracts were washed with brine, dried over anhydrous magnesium sulfateand filtered. The filtrate was concentrated under reduced pressure toprovide the title compound as a yellow oil (5.15 g, 82%) which was usedwithout further purification in Step D. ¹H-NMR: δ (CDCl₃, mixture ofsyn- and anti-isomers), 8.00 (1H, br s), 7.46 (0.6H, d, J=7.9 Hz),7.36–7.24 (5H, m), 6.80 (0.4H, d, J=7.0 Hz), 5.13 (0.8H, s), 5.09 (1.2H,s), 3.94 (0.4H, q, J=7.1 Hz), 3.27 (0.6H, td, J=6.4 and 8.0 Hz),1.94–1.58 (2H, m), 1.48–1.24 (4H, m) and 0.94–0.84 (3H, m).

Step D: 2RS-(Benzyloxyimino-methyl)-heptanoic acid(1S-dimethylcarbamoyl-2,2-dimethyl-propyl) amide

2-(Benzyloxyimino-methyl)-heptanoic acid (5.16 g, 20.7 mmol),tert-leucine N,N-dimethylamide (3.60 g, 22.77 mmol) and EDC (4.76 g,24.84 mmol) were stirred together in DMF (75 ml) and cooled to 0° C.HOAt (250 mg, cat.) was added and the bright yellow mixture was allowedto warm to room temperature and stirred overnight. The solvent wasremoved under reduced pressure and the residual oil was partitionedbetween ethyl acetate and 1M hydrochloric acid. The organic layer waswashed with brine, dried over anhydrous magnesium sulfate, filtered andconcentrated to dryness under reduced pressure. The title compound wasobtained as a colourless oil by flash chromatography (silica gel,gradient elution with 33% to 66% ethyl acetate in hexane). Yield 6.84 g(85%). ¹H-NMR: δ (CDCl₃, mixture of syn- and anti-isomers), 7.45 (0.6H,2d), 7.40–7.26 (5H, m), 6.72 (0.4H, 2d), 6.58 (1H, m), 5.20–4.69 (3H,m), 3.82 (0.4H, m), 3.16–3.10 (3H, m), 3.05 (0.6H, m), 2.99–2.92 (3H,m), 1.90–1.54 (2H, m), 1.39–1.17 (4H, m), 0.97 (2.7H, s), 0.96 (1.8H,s), 0.94 (2.7H, s), 0.92 (1.8H, s) and 0.92–0.82 (3H, m).

Step E: 2R (or S)-(Benzyloxyamino-methyl)-heptanoic acid(1S-dimethylcarbamoyl-2,2-dimethyl-propyl) amide

To a solution of 2RS-(benzyloxyimino-methyl)-heptanoic acid(1S-dimethyl-carbamoyl-2,2-dimethyl-propyl) amide (5.0 g, 12.84 mmol) inacetic acid (40 ml) was added sodium cyanoborohydride (2.02 g, 32.0mmol) in one portion. Over the course of 1 hour the reagent dissovedslowly with mild effervesence to give a colourless solution, which wasleft to stir overnight. The solvent was removed by evaporation underreduced pressure and azeotroping with toluene. The remaining oil waspartitioned between diethyl ether and 1 M sodium carbonate (CARE!-somegas evolved). The organic layer was washed with brine (70 ml), washedwith brine, dried over anhydrous magnesium sulfate, filtered andconcentrated to dryness under reduced pressure. The two diastereoisomersof title compound were purified and separated by flash chromatography(silica gel, gradient elution with 50% to 100% ethyl acetate in hexane).

Diastereoisomer A (faster eluting): colourless oil (2.27 g, 45%).¹H-NMR: δ (CDCl₃), 7.43–7.28 (5H, m), 6.76 (1H, br d, J=9.4 Hz), 5.69(1H, br s), 4.93 (1H, d, J=9.4 Hz), 4.72 (2H, s), 3.15 (3H, s),3.18–3.00 (2H, m), 2.96 (3H, s), 2.49 (1H, m), 1.66–1.49 (2H, m),1.46–1.19 (4H, m), 0.99 (9H, s) and 0.86 (3H, t, J=6.8 Hz).

Diastereoisomer B (slower eluting): colourless oil (1.44 g, 46%).¹H-NMR: δ (CDCl₃), 7.40–7.27 (5H, m), 6.70 (1H, br d, J=9.0 Hz), 5.99(1H, br s), 4.85 (1H, d, J=9.0 Hz), 4.71 (2H, d, J=1.6 Hz), 3.16 (3H,s), 3.06–2.97 (2H, m), 2.95 (3H, s), 2.57 (1H, m), 1.74–1.21 (6H, m),1.00 (9H, s) and 0.88 (3H, br t, J=6.7 Hz).

Step F: 2R (or S)-[(Benzyloxy-formyl-amino)-methyl]-heptanoic acid(1S-dimethylcarbamoyl-2,2-dimethyl-propyl) amide

2-(Benzyloxyamino-methyl)-heptanoic acid(1S-dimethylcarbamoyl-2,2-dimethyl-propyl) amide (diastereoisomer A)(2.02 g, 5.13 mmol) was dissolved in anhydrous THF (50 ml) and placedunder a blanket of argon. N-formyl-benzotriazole (A. R. Katritzky etal., Synthesis 1995, 503) (0.83 g, 5.65 mmol) was added and the mixturewas allowed to stir at room temperature for 4 hours. The solvent wasevaporated under reduced pressure and the residual oil was partitionedbetween dichloromethane and 1 M sodium hydroxide. The organic layer waswashed with more sodium hydroxide and brine, dried over anhydrousmagnesium sulfate, filtered and concentrated to dryness under reducedpressure. The title compound was obtained as a white crystalline solidby flash chromatography (silica gel, elution with 33% ethyl acetate inhexane). Yield 1.60 g (74%). ¹H-NMR: δ (CDCl₃, rotamers), 8.00 (1H, brm), 7.47–7.29 (5H, m), 6.25 (1H, br d, J=9.3 Hz), 5.08–4.74 (2H, br m),4.87 (1H, d, J=9.4 Hz), 3.89–3.52 (2H, br m), 3.13 (3H, s), 2.94 (3H,s), 2.54 (1H, m), 1.67–1.11 (6H, m), 0.95 (9H, s) and 0.85 (3H, br t,J=6.9 Hz).

2-(Benzyloxyamino-methyl)-heptanoic acid(1S-dimethylcarbamoyl-2,2-dimethyl-propyl) amide (diastereoisomer B) wassimilarly prepared from the the slower eluting diastereoisomer in StepE. Yield 0.38 g (41%). ¹H-NMR: δ (CDCl₃, rotamers), 8.00 (1H, br m),7.47–7.28 (5H, br m), 6.29 (1H, br d, J=9.3 Hz), 5.01–4.63 (2H, br m),4.88 (1H, d, J=9.3 Hz), 3.82–3.51 (1.5H, br m), 3.20–2.78 (6.5H, br m),2.50 (1H, br m), 1.76–1.17 (6H, br m), 0.97 (9H, s) and 0.85 (3H, br t,J=6.7 Hz).

Step G: 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-hexanoicacid-(1S-dimethyl carbamoyl-2,2-dimethyl-propyl)-amide

2-[(Benzyloxy-formyl-amino)-methyl]-heptanoic acid(1S-dimethylcarbamoyl-2,2-dimethyl-propyl) amide (diastereoisomer A)(1.43 g, 3.41 mmol) was dissolved in methanol (50 ml) and placed under ablanket of argon. A suspension of 10% palladium on charcoal (100 mg,cat.) in ethyl acetate (2 ml) was added and the mixture was stirredvigorously while hydrogen gas was bubbled through the solution. After 10minutes the mixture was placed under an atmosphere of hydrogen and leftto stir for 3 hours, whereupon TLC analysis indicated that all of thestarting material had been consumed. The system was purged with argonand the catalyst was removed by filtration. The filtrate wasconcentrated under reduced pressure to provide the title compound as acolourless hygroscopic foam (1.11 g, 99%). ¹H-NMR: δ (CDCl₃, rotamers),8.41 (0.35H, s), 7.83 (0.65H, br s), 6.80 (0.35H, br d, J=8.9 Hz), 6.62(0.65H, br d, J=9.4 Hz), 4.91 (0.65H, br d, J=9.4 Hz), 4.88 (0.35H, brd, J=8.9 Hz), 4.04 (1H, dd, J=14.7 and 7.4 Hz), 3.82 (0.65, dd, J=14.0and 9.7 Hz), 3.56 (0.35H, dd, J=14.7 and 3.3 Hz), 3.48 (0.65H, dd,J=14.0 and 4.0 Hz), 3.16 (1.05H, s), 3.15 (1.95H, s), 2.98 (1.05H, s),2.96 (1.95H, s), 2.90–2.74 (0.65H, br m), 2.74–2.61 (0.35H, br m)1.73–1.17 (6H, br m), 0.99 (3.15H, s). 0.95 (5.85H, s) and 0.87 (3H, brt, J=6.7 Hz). ¹³C-NMR; δ (CDCl₃), 174.6, 171.2, 162.2, 157.2, 60.1,54.5, 54.3, 52.3, 48.4, 44.8, 44.3, 35.6, 35.4, 29.6, 29.0, 26.3, 20.8,20.2, 14.0 and 13.7. LRMS: +ve ion 352 (M+Na), −ve ion 328 (M−H).

2-[(Formyl-hydroxy-amino)-methyl]-hexanoicacid-(1S-dimethylcarbamoyl-2,2-dimethyl-propyl)-amide (diastereoisomerB) was similarly prepared from diastereoisomer B in Step E ¹H-NMR; δ(CDCl₃, rotamers), 9.37 (0.5H, s), 8.40 (0.5H, s), 7.75 (0.5H, br s),6.62 (0.5H, br s), 6.41 (0.5H, br d, J=7.1 Hz), 4.87 (0.5H, br d, J=6.6Hz), 4.66 (0.5H, br d, J=7.6 Hz), 3.84–3.39 (2H, m), 3.21 (1.5H, br s),3.14 (1.5H, br s), 2.98 (3H, br s), 2.91–2.54 (1H, m), 1.79–1.23 (6H, m)and 1.08–0.83 (12H, m). ¹³C-NMR; δ (CDCl₃, rotamers), 174.9, 173.3,56.3, 54.8, 51.6, 50.3, 45.5, 45.1, 38.6, 38.4, 36.2, 36.0, 35.3, 34.4,29.5, 29.4, 29.3, 29.2, 26.6, 26.5, 22.6, 22.5 and 13.9. LRMS: +ve ion352 [M+Na], −ve ion 328 [M−H].

Method II

An alternative asymmetric synthetic route to the compound of Example 13is outlined in Scheme 3 and is described in detail below.

Step A: 2-Butyl acrylic acid

To a solution of n-butylmalonic acid (17.2 g, 107 mmol) in ethanol (200ml) was added piperidine (12.76 ml, 129 mmol) and 37% aq. formaldehyde(40.3 ml, 538 mmol). The solution was heated to 80° C. during which timea precipitate appeared and then gradually redissolved over 1 hour. Thereaction mixture was stirred at 80° C. overnight then cooled to roomtemperature. The solvents were removed under reduced pressure and theresidue was dissolved in ethyl acetate (200 ml), washed successivelywith 1 M hydrochloric acid and brine, dried over anhydrous magnesiumsulfate and filtered. The filtrate was concentrated to give the titlecompound as a clear oil (13.37 g, 97%). ¹H-NMR; δ (CDCl₃), 6.29 (1H, s),5.65 (1H, s), 2.34–2.28 (2H, m), 1.54–1.26 (4H, m) and 0.94 (3H, t,J=7.1 Hz).

Step B: 4S-Benzyl-3-(2-butyl-acryloyl)-5,5-dimethyl-oxazolidin-2-one

2-Butyl acrylic acid (21.5 g, 168 mmol) was dissolved in dry THF (500ml) and cooled to −78° C. under a blanket of argon. Triethylamine (30ml, 218 mmol) and pivaloyl chloride (21 ml, 168 mmol) were added at sucha rate that the temperature remained below −60° C. The mixture wasstirred at −78° C. for 30 minutes, warmed to room temperature for 2hours and finally cooled back to −78° C.

In a separate flask, 4S-benzyl-5,5-dimethyl-oxazolidin-2-one wasdissoved in dry THF (500 ml) and cooled to −78° C. under a blanket ofargon. n-Butyllithium (2.4 M solution in hexanes, 83 ml, 200 mmol) wasadded slowly and the mixture was stirred for 30 minutes at roomtemperature. The resulting anion was tranferred via a cannula into theoriginal reaction vessel. The mixture was allowed to warm to roomtemperature and was stirred overnight at room temperature. The reactionwas quenched with 1 M potassium hydrogen carbonate (200 ml) and thesolvents were removed under reduced pressure. The residue waspartitioned between ethyl acetate and water. The organic layer waswashed with brine, dried over anhydrous magnesium sulphate, filtered andconcentrated under reduced pressure to give an orange oil. TLC analysisrevealed the presence of unreacted chiral auxiliary in addition to therequired product. A portion of the material (30 g) was dissolved indichloromethane and flushed though a silica pad to give pure titlecompound as a yellow oil (25.3 g). ¹H-NMR;δ (CDCl₃), 7.31–7.19 (5H, m),5.41 (2H,s), 4.51 (1H, dd, J=9.7, 4.2 Hz), 3.32 (1H, dd, J=14.2, 4.2Hz), 2.82 (1H, dd, J=14.2, 9.7 Hz), 2.40–2.34 (2H, m), 1.48–1.32 (4H,m), 1.43 (3H, s), 1.27 (3H, s) and 0.91 (3H, t, J=7.1 Hz). Some chiralauxiliary was recovered by flushing the silica pad with methanol.

Step C:4S-Benzyl-3-[2-(benzyloxyamino-methyl)-hexanoyl]-5,5-dimethyl-oxazolidin-2-one(p-toluenesulfonic acid salt)

4S-Benzyl-3-(2-butyl-acryloyl)-5,5-dimethyl-oxazolidin-2-one (19.8 g,62.8 mmol) was mixed with O-benzylhydroxylamine (15.4 g, 126 mmol) andstirred overnight at room temperature. The mixture was dissolved inethyl acetate and the solution was washed with 1 M hydrochloric acid, 1M sodium carbonate and brine, dried over anhydrous magnesium sulfate andfiltered. The filtrate was concentrated under reduced pressure to a paleyellow oil (25.3 g) which was shown by NMR and HPLC analysis to contain4S-Benzyl-3-[2-(benzyloxyamino-methyl)-hexanoyl]-5,5-dimethyl-oxazolidin-2-one(ca. 82% d.e.) along with a trace of starting material. The product wascombined with another batch (26.9 g, 76% d.e.) and dissolved in ethylacetate (200 ml). p-Toluenesulfonic acid (22.7 g, 119 mmol) was addedand the mixture was cooled to 0° C. The title compound was obtained as awhite crytalline solid by seeding and scratching. Yield: 25.2 g, (34%,single diastereoisomer). A second crop (14.7 g, 20%, singlediastereoisomer) was also obtained. ¹H-NMR;δ (CDCl₃), 7.89 (2H, d, J=8.2Hz), 7.37–7.12 (10H, m), 7.02 (2H, d, J=6.9 Hz), 5.28–5.19 (2H, m), 4.55(1H, m), 4.23 (1H, m), 3.93 (1H, m), 3.58 (1H, m), 2.58 (1H, m), 2.35(3H, s), 1.67–1.51 (2H, m), 1.29–1.16 (4H, m), 1.25 (3H, s), 1.11 (3H,s) and 0.80–0.75 (3H, m).

Step D: 2R-Benzyloxyamino-methyl)-hexanoic acid

4S-Benzyl-3-[2R-(benzyloxyamino-methyl)-hexanoyl]-5,5-dimethyl-oxazolidin-2-onep-toluenesulfonic acid salt (25.2 g, 40.2 mmol) was partitioned betweenethyl acetate and 1 M sodium carbonate. The organic phase was dried overanhydrous magnesium sulfate, filtered and evaporated under reducedpressure. The residual oil was dissolved in THF (150 ml) and water (50ml) and cooled to 0° C. and treated with lithium hydroxide (1.86 g, 44.2mmol). The solution was stirred for 30 minutes at 0° C., then overnightat room temperature. The reaction was acidified to pH4 with 1 M citricacid and the solvents were removed. The residue was partitioned betweendichloromethane and 1 M sodium carbonate. The basic aqueous layer wasacidified to pH4 with 1M citric acid and extracted three times withethyl acetate. The combined organic layers were dried over anhydrousmagnesium sulfate, filtered and concentrated to provide the titlecompound as a colourless oil (7.4 g, 73%). ¹H-NMR;δ (CDCl₃), 8.42 (2H,br s), 7.34–7.25 (5H, m), 4.76–4.66 (2H, m), 3.20–3.01 (2H, m), 2.73(1H, m), 1.70–1.44 (2H, m), 1.34–1.22 (4H, m) and 0.92–0.86 (3H, m).

Step E: 2R-(Benzyloxyamino-methyl)-hexanoic acid(1S-dimethylcarbamoyl-2,2-dimethyl-1-propyl) amide

2R-Benzyloxyamino-methyl)-hexanoic acid (7.98 g, 31.8 mmol) wasdissolved in DMF (150 ml) and the solution was cooled to 0° C. EDC (6.1g, 31.8 mmol) and HOBt (430 mg, 3.2 mmol) were added and the mixture wasstirred for 15 minutes. tert-Leucine-N,N-dimethylamide (5.53 g, 34 mmol)was added and the reaction was allowed to warm to room temperature andwas stirred overnight. The solvent was removed under reduced pressureand the residue was dissolved in ethyl acetate, washed successively with1 M hydrochloric acid, saturated sodium hydrogen carbonate and brine,dried and filtered. The solvent was removed to leave the title compoundas a yellow oil (8.7 g, 69%) which was used in Step F without furtherpurification. ¹H-NMR; δ (CDCl₃), 7.35–7.28 (5H, m), 6.77 (1H, br d,J=9.2 Hz), 5.69 (1H, br s), 4.93 (1H, d, J=9.4 Hz), 4.72 (2H, s), 3.15(3H, s), 3.10–3.00 (2H, m), 2.95 (3H, s), 2.49 (1H, m), 1.64–1.21 (6H,m), 0.99 (9H, s) and 0.86 (3H, t, J=6.8 Hz).

Step F: 2R-[(Benzyloxy-formyl-amino)-methyl]-hexanoic acid(1S-dimethyl-carbamoyl-2,2-dimethyl-1-propyl) amide

2R-(Benzyloxyamino-methyl)-hexanoic acid(1S-dimethylcarbamoyl-2,2-dimethyl-1-propyl) amide (7.8 g, 19.9 mmol)was dissolved in dry THF (100 ml) and treated with1-formyl-benzotriazole (3.08 g, 21.0 mmol). The reaction was stirredovernight at room temperature. The solvent was removed under reducedpressure and the residue was dissolved in ethyl acetate, washed with 2 Msodium hydroxide solution and brine. The organic layer was dried overanhydrous magnesium sulfate, filtered and concentrated to dryness underreduced pressure. The product was crystallised from ether-hexane (4.83g, 57% in two crops). ¹H-NMR;δ (CDCl₃, rotamers), 8.12 (0.6H, br s),7.89 (0.4H, br s), 7.37 (5H, s), 6.25 (1H, d, J=9.3 Hz), 4.96 (0.6H, brs), 4.86 (1H, d, J=9.4 Hz), 4.80 (0.4H, br s), 3.74 (2H, br s), 3.13(3H, s), 2.94 (3H, s), 2.53 (1H, m), 1.38–1.21 (6H, m), 0.95 (9H, s) and0.85 (3H, t, J=6.9 Hz). Note: A small sample was crytallised fromether-hexane to provide crystals suitable for X-ray crystallography. Thestereochemistry was proven to be as stated herein.

Step G: 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-dimethylcarbamoyl-2,2-dimethyl-1-propyl) amide

2R-[(Benzyloxy-formyl-amino)-methyl]-hexanoic acid(1S-dimethylcarbamoyl-2,2-dmethyl-1-propyl) amide (4.72 g, 11.3 mmol)was dissolved in ethanol (80 ml) and placed under a blanket of argon. Asuspension of 10% palladium on charcoal (940 mg) in ethyl acetate (2 ml)was added and the mixture was stirred vigorously as hydrogen gas wasbubbled through the system. After 30 minutes the suspension was placedunder a balloon of hydrogen and stirred overnight at room temperature.The flask was purged with argon before removing the catalyst byfiltration. The filtrate was concentrated under reduced pressure toprovide the title compound as a colourless foam which crystallised onstanding (3.65 g, 98%). ¹H-NMR; δ (CDCl₃, rotamers), 9.32 (0.4H, br s),8.41 (0.4H, s), 7.88 (0.6H, br s), 7.27 (0.6H, s), 6.75 (0.4H, br d,J=8.8 Hz), 6.58 (0.6H, br d, J=9.3 Hz), 4.89 (1H, m), 4.04 (0.4H, m),3.82 (0.6H, m), 3.53 (1H, m), 3.16 (1.2H, s), 3.15 (1.8H, s), 2.98(1.2H, s), 2.96 (1.8H, s), 2.79 (0.6, m), 2.65 (0.4H, m), 1.78–1.58 (6H,m), 0.99 (3.6H, s), 0.95 (5.4H, s) and 0.87, 3H, t, J=6.7 Hz). ¹³C-NMR;δ(CDCl₃, rotamers), 175.8, 173.3, 172.0, 55.4, 54.9, 52.2, 48.8, 46.3,38.9, 38.8, 36.3, 36.1, 30.3, 30.2, 29.7, 26.9, 23.0 and 14.3. LRMS: +veion 352 [M+Na], −ve ion 328 [M−H].

The compounds of Examples 14 to 27 were prepared by analogy with Example13, Method I, substituting the appropriate ester for ethyl caproate inStep A. Where both diastereoisomers were prepared, diastereoisomer A isthe faster eluting and usually the more potent against PDF in vitro. Insome cases only the faster running diastereoisomer (Step E) was takenthrough to the final product.

EXAMPLE 14 2R (orS)-[(Formyl-hydroxy-amino)-methyl]-3-cyclopentyl-propionic acid(1S-dimethyl-carbamoyl-2,2-dimethyl-propyl)-amide

Diastereoisomer A. Colourless glass. ¹H-NMR; δ (CDCl₃, rotamers), 9.33(0.4H, br s), 8.94 (0.6H, br s), 8.40 (0.4H, s), 7.82 (0.6H, s), 6.82(0.4H, br d, J=8.6 Hz), 6.62 (0.6H, br d, J=9.3 Hz), 4.90 (1H, m), 4.06(0.4H, br dd, J=14.7, 7.3 Hz), 3.81 (0.6H, br dd, J=14.0, 9.7 Hz), 3.50(1H, m), 3.16 (1.2H, s), 3.14 (1.8H, s), 2.97 (1.2H, s), 2.95 (1.8H, s),2.80 (1H, m), 1.87–1.32 (9H, m), 1.16–0.95 (2H, m), 0.99 (3.6H, s) and0.95 (5.4H, s). ¹³C-NMR; δ (CDCl₃, rotamers), 172.9, 171.3, 55.0, 54.5,52.0, 48.6, 45.4, 44.2, 38.5, 38.4, 37.9, 37.6, 36.4, 36.3, 35.8, 35.6,35.5, 32.7, 32.6, 26.5, 26.4 and 25.1. LRMS: +ve ion 378 [M+Na], −ve ion354 [M−H].

Diastereoisomer B. Colourless glass. ¹H-NMR; δ (CDCl₃, rotamers), 9.30(0.6H, br s), 8.41 (0.6H, s), 7.75 (0.4H, s), 6.52 (0.4H, br d, J=8.7Hz), 6.41 (0.6H, br d, J=7.3 Hz), 4.85 (0.4H, br d, J=9.5 Hz), 4.63(0.6H, br d, J=7.5 Hz), 3.85–3.40 (2H, m), 3.25–2.95 (6H, 3br s), 2.78(1H, 2br m), 1.90–1.40 (8H, m), 1.30 (1H, m), 1.20–1.00 (2H, m) and1.05–0.95 (9H, 2s). ¹³C-NMR; δ (CDCl₃, rotamers), 174.9, 173.3, 172.8,56.5, 54.7, 51.5, 50.5, 44.7, 44.6, 38.6, 38.4, 38.0, 37.8, 36.2, 36.0,35.7, 35.5, 35.3, 34.3, 33.0, 32.9, 32.4, 32.3, 30.9, 26.6, 26.5, 25.1,25.0 and 24.9. LRMS: +ve ion 378 [M+Na], −ve ion 354 [M−H].

EXAMPLE 15 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-heptanoicacid-(1S-dimethylcarbamoyl-2,2-dimethyl-propyl)-amide

Diastereoisomer A. Dark orange oil. ¹H-NMR; δ (CDCl₃, rotamers), 8.32(0.33H, s), 7.76 (0.67H, br s), 6.78 (0.33H, br d, J=9.1 Hz), 6.68(0.67H, br d, J=9.1 Hz), 4.87–4.79 (1H, m), 3.96 (0.33H, br dd, J=14.6,7.6 Hz), 3.74 (0.67H, br dd, J=13.9, 9.7 Hz), 3.51–3.36 (1H, m), 3.09(1H, s), 3.08 (2H, s), 2.90 (1H, s), 2.89 (2H, s), 2.86–2.55 (1H, m),1.53–1.19 (8H, br m), 0.92 (3H, s), 0.88 (6H, s) and 0.79 (3H, m).¹³C-NMR; δ (CDCl₃, rotamers), 174.3, 172.0, 170.5, 170.4, 54.0, 53.5,53.4, 50.8, 49.7, 47.4, 44.9, 43.8, 37.5, 37.4, 34.8, 34.7, 34.6, 30.6,29.2, 29.1, 25.8, 25.5, 21.4 and 12.9. LRMS: +ve ion 344 M+H], −ve ion342 [M−H].

Diastereoisomer B. Dark orange oil. ¹H-NMR; δ (CDCl₃, rotamers), 8.36(0.5H, s), 7.74 (0.5H, s), 6.69 (0.5H, br s), 6.57 (0.5H, br d, J=7.6Hz), 4.89 (0.5H, br s), 4.70 (0.5H, d, J=7.8 Hz), 3.76–3.40 (2H, m),3.21 (1.5H, s), 3.16 (1.5H, s), 2.98 (3H, s), 2.81 (1H, br s), 2.72–2.60(1H, m), 1.67 (2H, br s), 1.42–1.22 (6H, m), 1.02 (4.5H, s), 0.99 (4.5H,s), 0.90 (1.5H, s) and 0.87 (1.5H, s). ¹³C-NMR; δ (CDCl₃, rotamers),175.2, 173.8, 173.1, 56.5, 55.1, 52.3, 51.1, 50.6, 45.8, 45.5, 39.0,38.9, 36.6, 36.3, 35.6, 34.9, 32.1, 32.0, 30.1, 29.9, 27.4, 27.4, 27.0,26.9, 22.9 and 14.3. LRMS: +ve ion 344 [M+H], −ve ion 342 [M−H].

EXAMPLE 16 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-pentanoic acid-(1S-dimethylcarbamoyl-2,2-dimethyl-propyl)-amide

Diastereoisomer A. White hygroscopic foam. ¹H-NMR; δ (CDCl₃, rotamers),8.40 (0.33H, s), 7.83 (0.67H, br s), 6.88 (0.33H, br d, J=8.6 Hz), 6.69(0.67H, br d, J=9.2 Hz), 4.90 (1H, t), 4.06 (0.33H, br dd, J=14.5, 7.4Hz), 3.82 (0.67H, br dd, J=13.7, 9.8 Hz), 3.57–3.44 (1H, m), 3.16 (1H,s), 3.15 (2H, s), 2.98 (1H, s), 2.96 (2H, s), 2.87–2.63 (1H, m),1.64–1.26 (4H, br m), 0.98 (3H, s), 0.94 (6H, s) and 0.90 (3H, t, J=7.3Hz). ¹³C-NMR; δ (CDCl₃, rotamers), 175.8, 173.2, 172.0, 55.4, 54.9,52.2, 48.7, 46.2, 45.0, 38.9, 38.9, 36.3, 36.1, 36.1, 32.7, 32.6, 27.0,26.9, 20.9, 20.8 and 14.4. LRMS: +ve ion 338 [M+Na], −ve ion 314 [M−H].

EXAMPLE 17 2R (or S)-[Formyl-hydroxy-amino)-methyl]-4-methyl-pentanoicacid-(1S-dimethyl-carbamoyl-2,2-dimethyl-propyl)-amide

Diastereoisomer A. White hygroscopic solid. ¹H-NMR; δ (CDCl₃, rotamers),8.41 (0.4H, s), 7.83 (0.6H, s), 6.65 (0.4H, d, J=8.6 Hz), 6.55 (0.6H, d,J=9.0 Hz), 4.91–4.83 (1H, m), 4.03–3.95 (0.4H, m), 3.84–3.74 (0.6H, m),3.62–3.43 (1H, m), 3.16 (1H, s), 3.13 (2H, s), 2.98 (1H, s), 2.96 (2H,s), 2.89–2.79 (0.6H, m), 2.76–2.71 (0.4H, m), 1.69–1.34 (1.8H, m),1.29–1.20 (1.2H, m), 1.0 (3.6H, s), 0.95 (5.4H, s) and 0.93–0.88 (6H,m). ¹³C-NMR; δ (CDCl₃, rotamers), 175.8, 173.3, 172.0, 171.7, 55.5,55.0, 52.4, 49.1, 44.3, 43.2, 39.5, 39.4, 38.9, 38.8, 36.3, 36.1, 27.0,26.9, 26.3, 26.0, 23.1, 23.0 and 22.8. LRMS: +ve ion 352 [M+Na], −ve ion328 [M−H].

EXAMPLE 18 3-Cyclohexyl-2R (orS)-[(formyl-hydroxy-amino)-methyl]-propionic acid(1S-dimethyl-carbamoyl-2,2-dimethyl-propyl)-amide

White solid. ¹H-NMR; δ (CDCl₃, rotamers), 8.38 (0.25H, s), 7.82 (0.75H,s), 6.93 (0.25H, d, J=8.9 Hz), 6.74 (0.75H, d, J=8.9 Hz), 4.90 (1H, d,J=9.4 Hz), 4.02 (0.25H, dd, J=9.7, 14.1 Hz), 3.78 (0.75H, dd, J=9.7,14.1 Hz), 3.46 (1H, m), 3.15 (3H, s), 2.96 (3H, s), 2.92 (1H, m), 1.65(6H, m), 1.20 (5H, m), 0.98 (9H, s) and 0.87 (2H, m). ¹³C-NMR; δ (CDCl₃,rotamers), 176.4, 174.2, 172.4, 56.0, 55.6, 53.4, 49.9, 44.0, 43.3,39.6, 39.4, 38.7, 38.5, 36.9, 36.7, 36.6, 34.8, 34.5, 27.5, 27.4 and27.2. LRMS: +ve ion 370 [M+H], 368 [M−H].

EXAMPLE 19 2R (or S)-Cyclopentyl-3-(Formyl-hydroxy-amino)-propionicacid-(1S-dimethylcarbamoyl-2,2-dimethyl-propyl)-amide

Diastereoisomer A. Off-white foam. ¹H-NMR; δ (CD₃OD, rotamers), 8.22(0.33H, s), 7.79 (0.66H, s), 4.89 (1H, s), 3.87 (1H, m), 3.50 (1H, m),3.19 (3H, s,), 2.93 (3H, s), 2.82 (0.66H, m), 2.65 (0.33H, m), 1.89 (2H,m), 1.56 (5H, m), 1.24 (2H, m) and 0.98 (9H, s). ¹³C-NMR; δ (CD₃OD,rotamers), 176.0, 56.7, 53.2, 51.1, 42.7, 39.2, 36.5, 36.4, 32.0, 27.4,26.4 and 26.2. IR (reflection disc) v_(max) 3318, 2953, 1663, 1628,1529, 1367, 1229, 1142, 1087, 877 cm⁻¹. LRMS: +ve ion 364 [M+Na], −veion 340 [M−H].

EXAMPLE 20 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-octanoic acid(1S-dimethylcarbamoyl-2,2-dimethyl-propyl)-amide

Diastereoisomer A. ¹H-NMR; δ (CDCl₃, rotamers), 8.40 (0.4H, s), 7.83(0.6H, s), 6.88 (0.4H, d, J=8.9 Hz), 6.68 (0.6H, d, J=9.2 Hz), 4.90 (1H,m), 4.05 (0.4H, m), 3.81 (0.6H, m), 3.50 (1H, m), 3.16 (1.2H, s), 3.15(1.8H, s), 2.97 (1.2H, s), 2.96 (1.8H, s), 2.86 (0.6H, m), 2.69 (0.4H,m), 1.59–1.25 (1OH, m), 1.14–0.95 (9H, m) and 0.89–0.77 (3H, m).¹³C-NMR; δ (CDCl₃, rotamers), 175.2, 172.9, 171.6, 171.4, 54.9, 54.5,54.3, 52.0, 48.4, 46.1, 45.7, 45.1, 44.7, 39.7, 38.5, 38.4, 35.8, 35.6,35.6, 31.7, 31.5, 30.2, 30.1, 29.1, 29.1, 27.0, 26.4, 22.4 and 14.0.LRMS: +ve ion 380 [M+Na], 358 [M+H], −ve ion 356 [M−H].

EXAMPLE 21 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-nonanoic acid(1S-dimethylcarbamoyl-2,2-dimethyl-propyl)-amide

Diastereoisomer A: brown solid. ¹H-NMR; δ (CDCl₃, rotamers), 9.30 (0.4H,s), 8.41 (0.6H, s), 7.83 (0.4H, s), 6.66 (0.4H, d, J=8.9 Hz), 6.52(0.6H, d, J=9.7 Hz), 4.92–4.84 (1H, m), 4.06–3.97 (0.4H, m), 3.87–3.77(0.6H, m), 3.63–3.45 (1H, m), 3.16 (1.2H, s), 3.14 (1.8H, s), 2.98(1.2H, s), 2.96 (1.8H, s), 2.86–2.74 (0.6H, m), 2.66–2.63 (0.4H, m),1.95–1.25 (12H, m), 1.00–0.95 (9H, m), and 0.90–0.84 (3H, m). ¹³C-NMR; δ(CDCl₃, rotamers), 175.5, 172.8, 171.4, 162.2, 156.1, 55.1, 54.5, 51.3,50.8, 48.4, 46.3, 44.9, 38.4, 38.4, 35.8, 35.7, 33.9, 31.7, 30.3, 30.2,29.4, 29.0, 27.1, 26.5, 26.5, 24.9, 22.6 and 14.0. LRMS: +ve ion 394[M+Na], 372 [M+H], −ve ion 370 [M−H].

EXAMPLE 22 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-decanoic acid(1S-dimethylcarbamoyl-2,2-dimethyl-propyl)-amide

Diastereoisomer A: colourless oil. LRMS: +ve ion 408 [M+Na], 386 [M+H],−ve ion 384 [M−H].

EXAMPLE 23 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-5-methyl-hexanoicacid (1S-dimethyl-carbamoyl-2,2-dimethyl-propyl)-amide

Diastereoisomer A: colourless oil. ¹H-NMR; δ (CDCl₃, rotamers), 9.31(0.4H, s), 8.40 (0.4H, s), 8.17 (0.6H, s), 6.77 (0.4H, d, J=7.5 Hz),6.60 (0.6H, d, J=8.0 Hz), 4.89 (1H, m), 4.04 (0.4H, m), 3.83 (0.6H, m),3.52 (1H, m), 3.16 (1.2H, s), 3.15 (1.8H, s), 2.98 (1.2H, s), 2.96(1.8H, s), 2.70 (1H, m), 1.58–1.14 (5H, m), 1.00–0.95 (9H, m) and0.87–0.84 (6H, m). ¹³C-NMR; δ (CDCl₃, rotamers), 172.9, 171.5, 162.2,156.3, 55.1, 54.6, 51.4, 48.5, 46.4, 45.0, 38.5, 38.4, 36.2, 35.9, 35.6,29.7, 28.1, 28.0, 27.9, 26.7, 26.6, 26.5 and 22.4. LRMS: +ve ion 366[M+Na], 344 [M+H], −ve ion 342 [M−H].

EXAMPLE 24 2R (or S)-[(Formyl-hydroxy-amino)-methyl]propanoic acid(1S-dimethylcarbamoyl-2,2-dimethyl-propyl)-amide

Diastereoisomer A: ¹H-NMR; δ (CDCl₃, rotamers), 8.41 (0.55H, s), 7.81(0.45H, s), 6.67 (0.45H, d, J=8.4 Hz), 6.51 (0.45H, d, J=7.2 Hz), 4.88(0.45H, d, J=9.4 Hz), 4.66 (0.55H, d, J=7.7 Hz), 3.76 (1H, m), 3.55(0.55H, dd, J=14.3, 9.8 Hz), 3.44 (0.45H, dd, J=14.2, 5.3 Hz), 3.21(1.65H, s), 3.14 (1.35H, s), 2.99 (1.65H, s), 2.97 (1.35H, s), 2.81 (1H,m), 1.21 (1.65H, d, J=6.7 Hz), 1.19 (1.35H, d, J=6.8 Hz), 1.01 (4.95H,s) and 0.98 (4.05H, s). LRMS: +ve ion 310 [M+Na], −ve ion 286 [M−H].

Diastereoisomer B: ¹H-NMR; δ (CDCl₃, rotamers), 9.47 (0.4H, br s), 8.41(0.4H, s), 7.86 (0.6H, s), 6.96 (0.4H, br s), 6.74 (0.6H, d, J=7.3Hz),4.91 (1H, m), 3.99 (0.4H, dd, J=14.2, 7.6 Hz), 3.83 (0.6H, dd, J=13.8,9.0 Hz), 3.50 (1H, m), 3.16 (1.2H, s), 3.15 (1.8H, s), 2.97 (3H, s),2.90 (1H, m), 1.21 (1.2H, d, J=6.8 Hz), 1.15 (1.8H, d, J=6.5 Hz), 0.99(3.6H, s) and 0.95 (5.4H, s). LRMS: +ve ion 310 [M+Na], −ve ion 286[M−H].

EXAMPLE 25 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-3-methyl butyricacid (1S-dimethyl-carbamoyl-2,2-dimethyl-propyl)-amide

Diastereoisomer A: ¹H-NMR; δ (CDCl₃, rotamers), 9.33 (0.4H, s), 8.38(0.4H, s), 7.81 (0.6H, s), 6.86 (0.4H, br s), 6.58 (0.6H, d, J=8.6 Hz),4.90 (1H, m), 4.06 (0.4H, dd, J=14.7, 7.3 Hz), 3.91 (0.6H, dd, J=13.8,10.6 Hz), 3.17 (1.2H, s), 3.15 (1.8H, s), 2.98 (1.2H, s), 2.96 (1.8H,s), 2.62 (0.6H, m), 2.48 (0.4H, m), 1.90 (1H, m), 1.09–0.86 (15H, m).LRMS: +ve ion 338 (M+Na), −ve ion 314 (M−H).

EXAMPLE 26 2R (orS)-[(Formyl-hydroxy-amino)-methyl]-3-phenyl-propionylpropionicacid-(1S-dimethylcarbamoyl-2,2-dimethyl-propyl)-amide

Diastereoisomer A. Colourless glass. ¹H-NMR; δ (CDCl₃, rotamers), 9.33(0.3H, br s), 8.95 (0.7H, br s), 8.43 (0.3H, br s), 7.83 (0.7H, br s),7.27–7.10 (5H, m), 6.65 (0.3H, br s), 6.45 (0.7H, br d, J=8.2 Hz),4.80–4.70 (1H, m), 4.22–4.10 (0.3H, m), 3.89 (0.7H, dd, J=13.7, 9.6 Hz),3.63–3.47 (1H, m), 3.20–2.69 (3H, m), 3.04 (3H, br s), 2.86 (3H, br s),and 0.87 (9H, br s). ¹³C-NMR; δ (CDCl₃, rotamers), 137.9, 137.7, 128.8,128.5, 126.6, 54.9, 54.5, 51.3, 48.3, 47.3, 46.6, 38.3, 38.2, 36.2,36.1, 35.8, 35.7, 35.6, 35.5 and 26.4. LRMS: +ve ion 386 (M+Na), −ve ion362 (M−H).

EXAMPLE 27 2R (orS)-[(Formyl-hydroxy-amino)-methyl]-3-(4-methoxy-phenyl)-propionicacid-(1S-dimethyl carbamoyl-2,2-dimethyl-propyl)-amide

Diastereoisomer A: LRMS: +ve ion 416 (M+Na), 394 (M+H), −ve ion 392(M−H).

The compounds of Examples 28 to 31 were prepared by analogy with Example13, Method II, substituting the appropriate amino acid amide or benzylester for tert-leucine N,N-dimethylamide in Step E.

EXAMPLE 28 2S-{2R-[Formyl-hydroxy-amino)-methyl]-hexanoylamino}-3-phenylpropionic acid

White foam. ¹H-NMR; δ (CD₃OD, rotamers), 8.11 (0.35H, s), 7.80 (0.65H,s), 7.31–7.16 (5H, m), 4.68 (1H, dd, J=8.7, 5.5 Hz), 3.58 (1H, m), 3.39(1H, m), 3.19 (1H, m), 2.98 (1H, m), 2.76 (1H, m), 1.55–1.26 (6H, m) and0.90–0.85 (3H, m). ¹³C-NMR; δ (CD₃OD, rotamers), 176.1, 175.7, 174.7,174.5, 138.6, 138.5, 130.3, 129.5, 129.4, 127.7, 55.0, 53.3, 49.8, 45.4,38.4, 38.3, 31.0, 30.8, 30.1, 23.7 and 14.2. IR (reflection disc)v_(max) 2932, 2359, 1727, 1660, 1551, 1454, 1381, 1221, 882, 701 cm⁻¹.LRMS: +ve ion 359 [M+Na], −ve ion 335 (M−H).

EXAMPLE 292S-{2R-[Formyl-hydroxy-amino)-methyl]-hexanoylamino}-3,3-dimethylbutyric acid

White foam. ¹H-NMR; δ (CD₃OD, rotamers), 8.25 (0.3H, s), 7.82 (0.7H, s),4.31 (1H, s), 3.83–3.29 (2H, m), 3.10–2.89 (1H, m), 1.54–1.33 (6H, m),1.03 (3H, s), 1.01 (6H, s) and 0.92–0.87 (3H, m). ¹³C-NMR; δ (CD₃OD,rotamers), 174.9, 172.9, 61.0, 52.4, 44.2, 44.0, 33.6, 30.1, 29.1, 26.2,22.6 and 13.1. IR(reflection disc) v_(max) 2959, 2359, 1644, 1537, 1371,1218, 881 and 704 cm⁻¹. LRMS: +ve ion 325 (M+Na), −ve ion 301 (M−H).

EXAMPLE 30 2S-[2R-(Formyl-hydroxy-amino)-methyl]-hexanoic acid{1-[(2S-hydroxymethyl-pyrrolidine-1-carbamoyl]-2,2-dimethyl-propyl}-amide

Colourless oil. ¹H-NMR; δ (CD₃OD, rotamers), 8.26 (0.4H, s), 7.84 (0.6H,s), 4.62 (0.4H, d, J=8.2 Hz), 4.39 (0.6H, d, J=8.4 Hz), 4.12 (1H, m),3.91–3.37 (6H, br m), 2.93 (0.6H, m), 2.78 (0.4H, m), 1.93 (5H, m), 1.45(2H, m), 1.39 (3H, m), 0.97 (3H, br s), 0.95 (3H, br s), and 0.89 (3H,t, J=6.7 Hz). ¹³C-NMR; δ (CDCl₃, rotamers), 174.8, 172.9, 65.3, 65.1,59.6, 59.5, 55.9, 55.7, 51.9, 47.8, 44.7, 44.0, 31.5, 30.5, 29.3, 28.7,28.1, 27.3, 23.8, 22.0, 21.2, 18.7, 18.3, 17.6, 14.7 and 13.3. LRMS: +veion 394 (M+Na), 372 (M+H), −ve ion 370 (M−H).

EXAMPLE 31 2S-[2R-(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1-[(2-hydroxy-ethyl)methyl-carbamoyl]-2,2-dimethyl-propyl}-amide

White foam. ¹H-NMR; δ (CD₃OD, rotamers), 8.25 (0.25H, s), 8.03 (0.125H,s), 7.82 (0.625H, s), 4.88 (1H, m), 3.83–3.54 (4H, br m), 3.41 (2H, m),3.25 (2H, s), 2.96 (2H, s and m), 1.49 (2H, m), 1.23 (4H, m), 1.00 (6H,s), 0.99 (3H, s), and 0.88 (3H, m). ¹³C-NMR; δ (CD₃OD, rotamers), 173.6,164.4, 61.1, 61.0, 56.9, 56.5, 54.2, 53.9, 52.2, 41.8, 38.9, 36.9, 36.3,35.3, 31.6, 30.8, 27.5, 24.1 and 14.7. LRMS: +ve ion 382 [M+Na], −ve ion358 [M−H].

The compounds of Examples 32 to 59 were prepared by analogy with Example7, Method II, substituting the appropriate amine or amino acidamide/benzyl ester for tert-leucine N,N-dimethylamide in Step E. In somecases HOAt was used in Step E and hydrogenolytic deprotection (Step G)was performed under catalytic transfer conditions (cyclohexene,palladium on charcoal in ethanol)

EXAMPLE 32 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoicacid-(1R-dimethylcarbamoyl-2,2-dimethyl-propyl)-amide

Colourless oil. LRMS: +ve ion 330 [M+H], −ve ion 328 [M−H].

EXAMPLE 33 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-dimethylcarbamoyl -2S-methyl-butyl)-amide

White foam. LRMS: +ve ion 352 [M+Na], −ve ion 328 [M−H].

EXAMPLE 34 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-dimethylcarbamoyl-2-methoxy-2-methyl-propyl)-amide

From racemic β-hydroxymethylvaline. Diastereoisomer A. Colourless oil.LRMS: +ve ion 368 [M+Na], 346 [M+H], −ve ion 344 [M−H]. DiastereoisomerB. LRMS: +ve ion 368 [M+Na], 346 [M+H], −ve ion 344 [M−H].

EXAMPLE 35 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-dimethylcarbamoyl-2-hydroxy-2-methyl-propyl)-amide

Colourless oil. LRMS: +ve ion 354 [M+Na], −ve ion 330 [M−H].

EXAMPLE 36 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid[2-(4-chloro-phenyl)-1S-dimethyl-carbamoyl-ethyl]-amide

Colourless oil. LRMS: +ve ion 330 (M+H), −ve ion 328 (M−H).

EXAMPLE 37 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid[1S-dimethylcarbamoyl-2-(4-hydroxy-phenyl)-ethyl]-amide

Colourless oil. LRMS: +ve ion 402 (M+Na), 380 (M+H).

EXAMPLE 38 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-dimethylcarbamoyl-2-naphthalen-2-yl-ethyl)-amide

Colourless oil. LRMS: +ve ion 414 (M+H), −ve ion 412 (M−H).

EXAMPLE 39 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(2-cyclohexyl-1S-dimethyl-carbamoyl-ethyl)-amide

White foam. LRMS: +ve ion 392 (M+Na), 370 (M+H)

EXAMPLE 40 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-dimethylcarbamoyl-phenyl-methyl)-amide

Colourless oil. LRMS: +ve ion 350(M+H), −ve ion 348 (M−H).

EXAMPLE 412-{2R-[(Formyl-hydroxy-amino)-methyl]-hexanoyl}-1,2,3,4-tetrahydro-isoquinoline-3S-carboxylicacid dimethylamide

LRMS: +ve ion 398 (M+Na), 376 (M+H), −ve ion 374 (M−H).

EXAMPLE 42 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(4-amino-1S-dimethylcarbamoyl-butyl)-amide

Colourless oil. LRMS: +ve ion 345 (M+H), −ve ion 343 (M−H).

EXAMPLE 43 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-dimethylcarbamoyl-2-hydroxy-ethyl)-amide

Colourless oil. LRMS: +ve ion 326 (M+Na), −ve ion 302 (M−H).

EXAMPLE 44N-Hydroxy-N-[2R-(4-methyl-piperazine-1-carbonyl)-hexyl]-formamide

LRMS: +ve ion 272 [M+H].

EXAMPLE 45 N-Hydroxy-N-[2R-(morpholine-4-carbonyl)-hexyl]-formamide

LRMS: +ve ion 281 (M+Na), 259 (M+H), −ve ion 257 (M−H).

EXAMPLE 46N-Hydroxy-N-[2R-(2S-hydroxymethyl-pyrrolidine-1-carbonyl)-hexyl]-formamide

LRMS: −ve ion 271 (M−H).

EXAMPLE 47 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-hydroxymethyl-2,2-dimethyl-propyl)-amide

LRMS: +ve ion 289 (M+H), −ve ion 287 (M−H).

EXAMPLE 48 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-methoxymethyl-2,2-dimethyl-propyl)-amide

LRMS: +ve ion 303 (M+H), −ve ion 301 (M−H).

EXAMPLE 49 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid[1S-(4-benzyl-piperidine-1-carbonyl)-2,2-dimethyl-propyl]-amide

LRMS: −ve ion 458 (M−H).

EXAMPLE 50 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid[1S-(benzyl-phenethyl-carbamoyl)-2,2-dimethyl-propyl]-amide

LRMS: +ve ion 496 (M+H), −ve ion 494 (M−H).

EXAMPLE 51 2S-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid[2,2-dimethyl-1S-(pyrrolidine-1-carbonyl)-propyl]-amide

LRMS: +ve ion 356 (M+H), −ve ion 354 (M−H).

EXAMPLE 52 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid[2,2-dimethyl-1S-(morpholine-4-carbonyl)-propyl]-amide

LRMS: +ve ion 372 (M+H), −ve ion 370 (M−H).

EXAMPLE 53 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid[2,2-dimethyl-1S-(4-methyl-piperazine-1-carbonyl)-propyl]-amide

LRMS: +ve ion 385 (M+H), −ve ion 383 (M−H).

EXAMPLE 54 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid[2,2-dimethyl-1S-(4-methyl-piperidine-1-carbonyl)-propyl]-amide

LRMS: +ve ion 384 (M+H), −ve ion 382 (M−H).

EXAMPLE 55 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-cyclohexylcarbamoyl-2,2-dimethyl-propyl)-amide

LRMS: +ve ion 398 (M+H), −ve ion 396 (M−H).

EXAMPLE 56 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid[1S-(4-acetyl-piperidine-1-carbonyl)-2,2-dimethyl-propyl]-amide

LRMS: +ve ion 412 (M+H), −ve ion 410 (M−H).

EXAMPLE 571-(2S-{2R-[(Formyl-hydroxy-amino)-methyl]-hexanoylamino}-3,3-dimethyl-butyryl)-piperidine-4-carboxylicacid methyl ester

LRMS: +ve ion 442 (M+H), −ve ion 440 (M−H).

EXAMPLE 58 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid[2,2-dimethyl-1S-(octahydro-quinoline-1-carbonyl)-propyl]-amide

LRMS: +ve ion 424 (M+H), −ve ion 422 (M−H).

EXAMPLE 59 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid[1S-(3,4-dihydro-2H-quinoline-1-carbonyl)-2,2-dimethyl-propyl]-amide

LRMS: −ve ion 416 (M−H).

EXAMPLE 602S-{3-Ethylsulfanymethyl-2R-[(formyl-hydroxy-amino)-methyl]propionylamino}-3,3,N,N-tetramethylbutyramide

A synthetic route to the title compound is outlined in Scheme 4 and isdescribed in detail below.

Step A: 2-Ethylsulfanylmethyl-acrylic acid

A mixture of malonic acid (5.2 g, 50 mmol), paraformaldehyde (3.3 g, 110mmol), dicyclohexylamine, 9.95 ml, 50 mmol) and ethanethiol 4.06 ml, 55mmol) in dioxane (120 ml) was heated at 70° C. for 2 hours. The solventswere removed under reduced pressure, the residue was redissolved inethyl acetate and the solution was extracted with saturated aqueoussodium hydrogen carbonate (4×20 ml). The combined aqueous layers werewashed with ethyl acetate (20 ml) then acidified with 1 M hydrochloricacid. The resulting suspension was extracted into dichloromethane andthe solution was dried over anhydrous magnesium sulfate, filtered andevaporated to provide the title compound as a white solid (3.76 g, 52%).¹H-NMR; δ (CDCl₃), 9.89 (1H, br s), 6.35 (1H, s), 5.77 (1H, s), 3.39(2H, s), 2.49 (2H, dd, J=7.4, 14.5 Hz) and 1.25 (3H, t, J=5.2 Hz).

Step B:4S-Benzyl-3-(2-ethylsulfanylmethyl-acryloyl)-5,5-dimethyl-oxazolidin-2-one

2-Ethylsulfanylmethyl-acrylic acid (3.76 g, 25.8 mmol) was dissolved indry THF (75 ml) and cooled to −78° C. under a blanket of argon.Triethylamine (4.6 ml, 33.5 mmol) and pivaloyl chloride (3.17 ml, 25.8mmol) were added at such a rate that the temperature remained below −60°C. The mixture was stirred at −78° C. for 30 minutes, warmed to roomtemperature for 2 hours and finally cooled back to −78° C.

In a separate flask, 4S-benzyl-5,5-dimethyl-oxazolidin-2-one wasdissoved in dry THF (75 ml) and cooled to −78° C. under a blanket ofargon. n-Butyllithium (2.4M solution in hexanes, 12.9 ml, 30.9 mmol) wasadded slowly and the mixture was stirred for 30 minutes at roomtemperature. The resulting anion was tranferred via a cannula into theoriginal reaction vessel. The mixture was allowed to warm to roomtemperature and stirred overnight at room temperature. The reaction wasquenched with saturated sodium hydrogen carbonate (20 ml) and thesolvents were removed under reduced pressure. The residue waspartitioned between ethyl acetate and water. The organic layer waswashed successively with saturated sodium hydrogen carbonate, 1 Mhydrochloric acid and brine, dried over anhydrous magnesium sulfate,filtered and concentrated under reduced pressure. The residue waspurified by flash chromatography (silica gel, 20% ethyl acetate inhexane) to provide the title compound as a yellow oil (6.5 g, 76%).¹H-NMR; δ (CDCl₃), 7.29 (5H, m), 5.58 (1H, s), 5.49 (1H, s), 4.54 (1H,dd, J=3.9, 9.7 Hz), 3.52 (2H, dd, J=15.8, 3.1 Hz), 3.38 (1H, dd, J=3.9,14.5 Hz), 2.84 (1H, dd, J=4.6, 14.3 Hz), 2.52 (2H, dd, J=7.2, 14.6 Hz),1.42 (3H, s), 1.29 (3H, s) and 1.22 (3H, t, J=7.5 Hz). LRMS: +ve ion 356(M+Na), 334 (M+H).

Step C:4S-Benzyl-3-[(2R-tert-butoxyamino-methyl)-3-ethylsulfanylmethyl-propionyl]-5,5-dimethyl-oxazolidi-2-one

4S-Benzyl-3-(2-ethylsulfanylmethyl-acryloyl)-5,5-dimethyl-oxazolidin-2-one(2.1 g, 6.3 mmol) was dissolved in ethanol (10 ml) andO-tert-butyl-hydroxylamine hydrochloride (0.95 g, 7.56 mmol) was added,followed by triethylamine (1.1 ml, 7.87 mmol). The mixture was stirredat 30° C. overnight. The solvents were removed under reduced pressureand the residue was dissolved in ethyl acetate. The organic solution waswashed succesively with 1 M hydrochloric acid, saturated sodium hydrogencarbonate and brine, dried over anhydrous magnesium sulphate andfiltered. The filtrate was concentrated under reduced pressure toprovide the title compound as a pale yellow oil (2.42 g, 91%; singlediastereoisomer by HPLC). ¹H-NMR; δ (CDCl₃), 7.30 (5H, m), 5.09 (1H, brs), 4.54 (1H, dd, J=3.5, 9.9 Hz), 4.33 (1H, m), 3.19 (2H, m), 3.08 (1H,dd, J=5.4, 11.8 Hz), 2.80 (3H, m), 2.56 (2H, dd, J=7.4, 14.7 Hz), 1.41(3H, s), 1.36 (3H, s), 1.23 (3H, t, J=7.3 Hz) and 1.13 (9H, s). LRMS:+ve ion 423 (M+H).

Step D: (2R-tert-butoxyamino-methyl)-3-ethylsulfanylmethyl-propionicacid

A solution of4S-Benzyl-3-[(2R-tert-butoxyamino-methyl)-3-ethylsulfanylmethyl-propionyl]-5,5-dimethyl-oxazolidin-2-onein (2.42 g, 5.72 mmol) THF (40 ml) was cooled to 0° C. and a solution oflithium hydroxide (288 mg, 6.86 mmol) in water (10 ml) was added. Themixture was allowed to warm to room temperature then stirred for 5hours. The solvent was removed under reduced pressure and the residuewas partitioned between water and ethyl acetate. The aqueous layer wasremoved and the ethyl acetate layer was washed successively with waterand saturated sodium hydrogen carbonate. The combined aqueous layerswere washed with ethyl acetate (20 ml) before acidifying with 1 Mhydrochloric acid. The resulting emulsion was extracted withdichloromethane (3×20 ml) and the combined organic layers were driedover anhydrous magnesium sulfate, filtered and evaporated to provide thetitle compound as a colourless oil (0.68 g, 50%). ¹H-NMR; δ (CDCl₃),8.03 (2H, br s), 3.21 (2H, d, J=6.1 Hz), 2.89 (2H, m), 2.75 (1H, m),2.57 (2H, dd, J=7.4, 14.8 Hz), 1.26 (3H, t, J=7.4 Hz) and 1.18 (9H, s).LRMS: +ve ion 236 [M+H], −ve ion 234 M−H].

Step E: A solution of2S-[2R-(tert-butoxy-amino-methyl)-3-ethylsulfanylmethyl-propionylamino}-3,3,N,N-tetramethylbutyramide

2R-tert-butoxyamino-methyl)-3-ethylsulfanylmethyl-propionic acid (340mg, 1.44 mol) was dissolved in DMF (10 ml) andtert-leucine-N,N-dimethylamide (272 mg, 1.73 mmol), HOAt (19.6 mg, 0.14mmol) and EDC (331 mg, 1.73 mmol) were added. The reaction was stirredovernight at room temperature. The solvent was removed under reducedpressure and the residue was dissolved in dichloromethane. The organicsolution was washed successively with 1 M hydrochloric acid, 1 M sodiumcarbonate and brine, dired over anhydrous magnesium sulfate andfiltered. The filtrate was concentrated under reduced pressure toprovide the required product as a colourless oil (440 mg, 82%). ¹H-NMR;δ (CDCl₃), 6.87 (1H, d, J=9.0 Hz), 5.11 (1H, br s), 4.93 (1H, d, J=9.3Hz), 3.15 (3H, s), 3.11 (1H, m), 2.95 (3H, s), 2.79 (3H, m), 2.54 (3H,s), 1.22 (3H, t, J=7.6 Hz), 1.18 (9H, s) and 1.01 (9H, s). LRMS: +ve ion398 [M+Na], 376 [M+1].

Step F:2S-{2R-[(tert-Butoxy-formyl-amino)-methyl]-3-ethylsulfanylmethyl-propionylamino}-3,3,N,N-tetramethylbutyramide

A solution of2S-[2R-(tert-butoxy-amino-methyl)-3-ethylsulfanylmethyl-propionylamino}-3,3,N,N-tetramethylbutyramide (220 mg, 0.58 mmol) indichlormethane (5 ml) was cooled to 0° C. and treated with formic aceticanhydride (0.1 ml). The reaction was stirred at room temperature for 4hours, then the solvent was evaporated under reduced pressure. Theresidue was purified by flash chromatography (silica gel, 50% ethylacetate in hexane as eluent) to provide the title compound as acolourless oil (120 mg, 52%). ¹H-NMR; δ (CDCl₃, rotamers), 8.31 (1H, brs), 6.56 (1H, d, J=9.1 Hz), 4.94 (0.33H, d, J=9.4 Hz), 4.88 (0.67H, d,J=9.2 Hz), 4.08 (0.67H, br m), 3.83 (1.34H, br m), 3.13 (3H, s), 2.95(3H, s), 2.80 (2H, m), 2.61 (1H, dd, J=6.8, 14.0 Hz), 2.49 (2H, dd,J=7.4, 14.7 Hz), 1.29 (9H, s), 1.25 (3H, t, J=7.2 Hz) and 0.99 (9H, s).LRMS: +ve ion 426 [M+Na], 404 [M+H].

Step G:2S-{3-Ethylsulfanymethyl-2R-[(formyl-hydroxy-amino)-methyl]propionylamino}-3,3,N,N-tetramethylbutyramide

A solution of2S-{2R-[(tert-butoxy-formyl-amino)-methyl]-3-ethylsulfanylmethyl-propionylamino}-3,3,N,N-tetramethylbutyramide (120 mg, 0.3 mmol) indeuterochloroform (1 ml) was treated with TFA (4 ml) and allowed tostand at 4° C. overnight. The solvents were removed under reducedpressure and residual TFA was removed by azeotroping with toluene. Theresidue was purified by preparative HPLC to provide the title compoundas a colourless oil (40 mg, 38%; 7:2 mixture of diastereomers by HPLC).¹H-NMR; δ (CDCl₃, rotamers), 8.40 (0.33H, s), 7.87 (0.67H, s), 7.24(0.33H, d, J=9.3 Hz), 6.98 (0.67H, d, J=9.3 Hz), 4.91 (0.67H, d, J=9.3Hz), 4.90 (0.33H, d, J=9.3 Hz), 4.07 (0.33H, dd, J=7.5, 14.5 Hz), 3.86(0.67H, dd, J=8.8, 14.2 Hz), 3.75 (0.67H, m), 3.68 (0.33H, m), 3.16 (1H,s), 3.15 (2H, s), 3.05 (1H, m), 2.96 (3H, s), 2.77 (1H, m), 2.66 (1H,m), 2.52 (2H, dd, J=7.4, 14.8 Hz), 1.22 (3H, t, J=7.3 Hz), 0.99 (3H, s)and 0.96 (6H, s). ¹³C-NMR; δ (CDCl₃, rotamers), 173.3, 171.6, 171.2,55.2, 54.8, 51.1, 48.5, 45.2, 44.4, 38.5, 38.4, 35.9, 35.8, 35.7, 31.7,31.4, 26.7, 26.6, 26.5 and 14.6. LRMS: +ve ion 370 [M+Na], 348 [M+H],−ve ion 346 [M−H].

The compound of Example 61 was prepared similarly using piperidine inplace of ethanethiol in Step A.

EXAMPLE 612-{2-[(Formyl-hydroxy-amino)-methyl]-3-piperidin-1-yl-propionylamino}-3,3,N,N-tetramethyl-butyramide

White solid (4:1 mixture of diastereoisomers by HPLC). ¹H-NMR; δ (CDCl₃,rotamers), 8.29 (1H, s), 7.95 (1H, br s), 4.87 (1H, d, J=9.1 Hz), 4.02(1H, dd, J=5.0, 14.6 Hz), 3.56 (1H, dd, J=8.2, 14.6 Hz), 3.14 (3H, s),2.96 (3H, s), 2.89 (1H, m), 2.69 (1H, m), 2.52 (5H, m), 1.65 (4H, m),1.49 (2H, m) and 0.99 (9H, s). ¹³C-NMR; δ (CDCl₃), 172.2, 171.3, 60.4,55.0, 54.9, 48.6, 42.4, 38.8, 36.2, 36.1, 27.0, 25.6 and 24.3. LRMS: +veion 371 [M+H], −ve ion 369 [M−H].

The compounds of Examples 62 to 65 were prepared by analogy with Example7, Method II, substituting O-tert-butylhydroxylamine forO-benzylhydroxylamine in Step B and the appropriate amine or amino acidamide/benzyl ester for tert-leucine N,N-dimethylamide in Step E. Finaldeprotection was performed by acidolysis with TFA (see Example 60,above).

EXAMPLE 62 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1R-dimethylcarbamoyl-2-methyl-2-methylsulfanyl-propyl)-amide

Colourless oil. ¹H-NMR; δ (CDCl₃, rotamers), 8.4 (0.5H, s), 7.85 (0.5H,s), 7.11 (0.5H, d, J=9.1 Hz), 6.93 (0.5H, d, J=9.1 Hz), 5.15 (1H, d,J=9.4 Hz), 3.90 (0.5H, m), 3.73 (0.5H, m), 3.64 (0.5H, d, J=14.3 Hz),3.48 (0.5H, dd, J=14.0, 3.9 Hz), 3.22 (3H, s), 2.97 (3H, s), 2.83 (0.5H,m), 2.70 (0.5H, m), 2.07 (1.5H, s), 2.04 (1.5H, s), 1.58 (1H, m), 1.36(4H, m), 1.32 (3H, s), 1.28 (3H, s) and 0.86 (3H, t, J=6.6 Hz). ¹³C-NMR;δ (CDCl₃, rotamers), 175.4, 173.5, 170.8, 63.6, 53.2, 53.1, 52.5, 49.5,47.5, 46.1, 44.9, 41.6, 37.5, 36.5, 36.4, 35.4, 30.2, 29.8, 28.0, 14.3,12.0 and 11.9. LRMS: +ve ion 362 [M+H], −ve ion 360 [M−H].

EXAMPLE 63 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(2-benzylsulfanyl-1R-dimethyl-carbamoyl-2-methyl-propyl)-amide

White foam. ¹H-NMR; δ (CDCl₃, rotamers), 8.37 (0.33H, s), 7.81 (0.66H,s), 7.31 (5H, m), 7.06 (0.33H, d, J=8.8 Hz), 6.89 (0.66H, d, J=9.3 Hz),5.20 (1H, d, J=9.3 Hz), 3.94 (0.33H, dd, J=8.3, 14.6 Hz), 3.78 (2.66H,m), 3.61 (0.33H, dd, J=3.5, 14.4 Hz), 3.42 (0.66H, dd, J=5.1, 14.9 Hz),3.21 (3H, s), 3.03 (3H, s), 2.82 (0.66H, m), 2.69 (0.33H, m), 1.61 (1H,m), 1.42 (1H, m), 1.37 (3H, s), 1.32 (3H, s), 1.26 (4H, m) and 0.86 (3H,t, J=6.6 Hz). ¹³C-NMR; δ (CDCl₃, rotamers), 175.3, 173.5, 171.0, 138.1,137.4, 129.5, 129.3, 129.1, 129.0, 128.9, 127.6, 127.4, 55.9, 53.7,52.5, 51.2, 49.6, 49.5, 46.1, 44.9, 39.0, 38.6, 36.6, 36.4, 33.9, 33.7,30.3, 30.1, 29.7, 26.7, 26.1, 25.7, 25.5, 24.2, 22.9 and 14.3. LRMS: +veion 460 [M+Na], 438 [M+H], −ve ion 436 [M−H].

EXAMPLE 64 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid[2-benzylsulfanyl-2-methyl-1R-(morpholine-4-carbonyl)-propyl]-amide

White foam. ¹H-NMR; δ (CDCl₃, rotamers), 8.44 (0.5H, s), 8.37 (0.5H, s),7.30 (5H, m), 6.88 (0.5H, d, J=8.3 Hz), 6.78 (0.5H, d, J=9.2 Hz), 5.12(1H, d, J=9.5 Hz), 3.91 (1H, dd, J=8.2, 14.6 Hz), 3.78 (10H, m), 3.45(1H, dd, J=4.5, 14.2 Hz), 2.80 (0.5H, m), 2.64 (0.5H, m), 1.61 (1H, m),1.41 (1H, m), 1.36 (3H, s), 1.33 (3H, s), 1.29 (4H, m) and 0.87 (3H, t,J=6.8 Hz). ¹³C-NMR; δ (CDCl₃, rotamers), 175.5, 173.4, 169.4, 137.8,129.5, 129.3, 129.1, 129.0, 127.8, 127.5, 67.1, 67.0, 53.3, 53.2, 51.99,49.6, 49.5, 49.2, 47.9, 46.5, 45.0, 43.2, 43.0, 34.0, 30.3, 30.2, 29.7,26.8, 26.5, 25.9, 25.8, 22.9 and 14.3. LRMS: +ve ion 502 [M+Na], 480[M+H], −ve ion 478 [M−H].

EXAMPLE 65 2-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid[2-benzylsulfanyl-2-methyl-1R (orS)-(4-methyl-piperidine-1-carbonyl)-propyl]-amide

Diastereoisomer A. White solid. LRMS: +ve ion 514 [M+Na], 492 [M+H], −veion 490 [M−H].

Diastereoisomer B. Colourless gum. LRMS: +ve ion 514 [M+Na], 492 [M+H],−ve ion 490 [M−H].

The compounds of Examples 66 to 68 were prepared by analogy with Example7, Method II, substituting the appropriate malonic acic for butylmalonicacid in Step A. O-tert-butylhydroxylamine for O-benzylhydroxylamine inStep C. Stereoselectivity in the Michael addition was variable. Finaldeprotection was performed by acidolysis with TFA (see Example 60,above).

EXAMPLE 66 2R-[(Formyl-hydroxy-amino)-methyl]-pent-4-enoic acid(1S-dimethylcarbamoyl-2,2-dimethyl-propyl)-amide

Single diastereoisomer. ¹H-NMR; δ (CDCl₃, rotamers), 8.40 (0.25H, s),7.84 (0.75H, s), 7.05 (0.35H, d, J=9.0 Hz), 6.74 (0.65H, d, J=9.3 Hz),5.70 (1H, m), 5.03–5.24 (2H, m), 4.88 (1H, dd, J=9.4, 6.7 Hz), 3.98(0.5H, m), 3.81 (0.5H, m), 3.55 (1H, m), 3.14 (3H, s), 2.97 (1.3H, s),2.96 (1.7H, s), 2.75–2.92 (1H, m), 2.16–2.50 (2H, m), 0.98 (4.5H, s) and0.94 (4.5H, s). LRMS: +ve ion 336 [M+Na], −ve ion 312 [M−H].

EXAMPLE 67 2R-[(Formyl-hydroxy-amino)-methyl]-hex-5-enoic acid(1S-dimethylcarbamoyl-2,2-dimethyl-propyl)-amide

Diastereoisomer A: colourless oil. ¹H-NMR; δ (CDCl₃, rotamers), 8.42(0.45H, s), 7.84 (0.55H, s), 6.78 (0.45H, d, J=8.4 Hz), 6.60 (0.55H, d,J=9.3 Hz), 5.74 (1H, m), 5.03 (2H, m), 4.88 (1H, m), 4.14 (0.4H, m),3.81 (0.6H, m), 3.55 (1H, m), 3.16 (1H, s), 3.15 (2H, s), 2.98 (1H, s),2.97 (2H, s), 2.85 (0.7H, m), 2.68 (0.3H, m), 2.07 (2H, m), 1.73 (1.6H,m), 1.50 (0.4H, m), 0.99 (4H, s) and 0.95 (5H, s). LRMS: +ve ion 350[M+Na], −ve ion 326 [M−H].

Diastereoisomer B: colourless oil. ¹H-NMR; δ (CDCl₃, rotamers), 8.41(0.5H, s), 7.75 (0.5H, s), 6.58 (0.5H, d, J=9.1 Hz), 6.36 (0.5H, d,J=9.1 Hz), 5.75 (1H, m), 5.01 (2H, m), 4.86 (0.5H, d, J=9.5 Hz), 4.64(0.5H, d, J=7.5 Hz), 3.42–3.82 (2H, m), 3.22 (1.5H, s), 3.07 (1.5H, s),2.99 (3H, s), 2.87 (0.5H, m), 2.66 (0.5H, m), 2.13 (2H, m), 1.81 (1H,m), 1.49 (1H, m), 1.02 (4.5H, s) and 1.00 (4.5H, s). LRMS: +ve ion 350[M+Na], −ve ion 326 [M−H].

EXAMPLE 68 2R-[(Formyl-hydroxy-amino)-methyl]-hex-4-ynoic acid(1S-dimethylcarbamoyl-2,2-dimethyl-propyl)-amide

Diastereoisomer A: colourless oil. ¹H-NMR; δ (CDCl₃, rotamers), 8.39(0.4H, s), 7.87 (0.6H, s), 7.20 (0.4H, d, J=8.4 Hz), 6.94 (0.6H, d,J=9.3 Hz), 4.90 (1H, m), 3.66–4.14 (2H, m), 3.16 (2H, s), 3.14 (2H, s),2.96 (3H, s), 2.88 (1H, m), 2.41 (2H, m), 1.77 (3H, m), 1.00 (3.5H, s)and 0.96 (5.5H, s). LRMS: +ve ion 348 [M+Na], −ve ion 324 [M−H].

Diastereoisomer B: Colourless oil. ¹H-NMR; δ (CDCl₃, rotamers), 8.37(0.5H, s), 7.81 (0.5H, s), 6.87 (1H, m), 4.91 (0.5H, d, J=9.4 Hz), 4.79(0.5H, d, J=8.2 Hz), 3.76 (1.5H, m), 3.63 (0.5H, m), 3.19 (1.5H, s),3.14 (1.5H, s), 2.98 (3H, s), 2.85 (1H, s), 2.41 (2H, m), 1.77 (3H, m),1.03 (4.5H, s) and 1.01 (4.5H, s). LRMS: +ve ion 348 [M+Na], −ve ion 324[M−H].

EXAMPLE 69 2R-[1R (or S)-(Formyl-hydroxy-amino)-ethyl]-hexanoic acid(1S-dimethylcarbamoyl-2,2-dimethyl-propyl)-amide

The title compound was prepared according to the route outlined inScheme 5 and as described in detail below:

Step A: 4-Benzyl-3-hexanoyl-oxazolidin-2-one

4S-Benzyl-oxazolidin-2-one (14.5 g, 81.7 mmol) was dissolved in dry THF(75 ml) under an argon atmosphere. The solution was cooled in an icebath before slow addition of n-butyllithium (1.6 M in hexanes, 56 ml,89.2 mmol). The lithium salt crystallised from the solution as a solidmass and was allowed to warm to room temperature overnight. Theresulting orange suspension was cooled again in an ice bath during theaddition of a cold solution of hexanoyl chloride (10.4 ml, 74.3 mmol) indry THF (50 ml). The mixture was left to warm to room temperature andwas then stirred for 3 hours. The reaction was quenched with 1M sodiumcarbonate solution (5 ml) and the solvent was removed under reducedpressure. The residue was partitioned between 1 M sodium carbonate (100ml) and ethyl acetate (150 ml). The organic layer was removed and theaqueous layer was extracted with more ethyl acetate. The combinedorganic layers were washed successively with water, 1 M sodium carbonateand brine, dried over anhydrous magnesium sulphate and filtered. Thefiltrate was concentrated to leave an orange oil. Purification by flashchromatography afforded the title compound as a yellow oil (10.21 g,50%). ¹H-NMR; δ (CDCl₃), 7.38–7.24 (3H, m), 7.24–7.16 (2H, m), 4.68 (1H,m), 4.24–4.12 (2H, m), 3.30 (1H, dd, J=13.4, 3.2 Hz), 3.02–2.86 (2H, m),2.77 (1H, dd, J=13.4, 9.6 Hz), 1.77–1.63 (2H, m), 1.44–1.30 (4H, m) and0.92 (3H, br t, J=6.9 Hz).

Step B: 1-(4S-Benzyl-2-oxo-oxazolidin-3-yl)-2R-butyl-butane-1,3-dione

4-Benzyl-3-hexanoyl-oxazolidin-2-one (10.2 g, 37.1 mmol) was dissolvedin THF (150 ml) under an argon atmosphere and cooled to −78° C. Lithiumhexamethyldisilazide (1 M in THF, 41 ml, 41 mmol) was added via acannula over a few minutes and the resulting green solution was stirredat −78° C. for 2 hours. Acetyl chloride (3.3 ml, 46.3 mmol) was addedslowly and the reaction mixture was stirred for 3.5 hours. A solution ofcitric acid (3.0 g, 14 mmol) in water (15 ml) was added quickly toquench the reaction. The solvent was removed under reduced pressure andthe residue was partitioned between ethyl acetate and water, washed withbrine, dried over anhydrous magnesium sulphate and filtered. Thefiltrate was concentrated to provide the title compound as a yellow oil(12.11 g, contains residual solvent) which was used without furtherpurification in Step C. ¹H-NMR; δ (CDCl₃), 7.37–7.21 (5H, m), 4.68 (1H,m), 4.53 (1H, dd, J=9.6, 3.7 Hz), 4.23–4.13 (2H, m), 3.43 (1H, dd,J=13.5, 3.3 Hz), 2.75 (1H, dd, J=13.5, 9.9 Hz), 2.33 (3H, s), 2.03 (1H,m), 1.77 (1H, m), 1.46–1.26 (4H, m) and 0.98–0.86 (3H, m).

Step C: 1-(4S-Benzyl-2-oxo-oxazolidin-3-yl)-2R-butyl-butane-1,3-dione3-(O-benzyl-oxime)

To a solution of1-(4S-benzyl-2-oxo-oxazolidin-3-yl)-2R-butyl-butane-1,3-dione (12.11 g,38.15 mmol) in water (10 ml) and ethanol (90 ml) was added sodiumacetate (3.75 g, 45.78 mmol) and O-benzyl hydroxylamine hydrochloride(7.31 g, 45.78 mmol). The resulting suspension was left to stir at roomtemperature overnight. The product (7.3 g, 45%, single oxime isomer)crystallised directly from the reaction and was filtered, washed withaqueous ethanol (1:1) and dried under vacuum. Further material (5.31 g,33%, mixture of oxime isomers) was obtained as a yellow oil from themother liquors by acid-base extraction followed by columnchromatography. ¹H-NMR; δ (CDCl₃, major oxime isomer), 7.34–7.20 (8H,m), 7.12–7.07 (2H, m), 5.14–5.02 (2H, m), 4.53 (1H, m), 4.13 (1H, dd,J=9.4, 4.0 Hz), 4.04 (1H, br t, J=8.4 Hz), 3.91 (1H, dd, J=9.0, 2.7 Hz),3.16 (1H, dd, J=13.4, 2.9 Hz, 2.09 (3H, s), 1.97 (1H, m), 1.75 (1H, dd,J=13.4, 10.8 Hz), 1.67 (1H, m), 1.45–1.22 (4H, m) and 0.91 (3H, br t,J=6.9 Hz).

Step D: 4S-Benzyl-3-[2R-(1R (orS)-benzyloxyamino-ethyl)-hexanoyl]-oxazolidin-2-one

The mixture of oximes form Step C (5.31 g, 12.5 mmol) was dissolved inacetic acid (30 ml) and cooled in an ice-water bath before addition ofsodium cyanoborohydride (0.8 g, 12.5 mmol) in one portion. Effervescencesubsided after a few minutes and a a further portion of borohydride (0.8g) was added. The reaction was allowed to warm to room temperature andstirred overnight. The acetic acid was removed under reduced pressureand the residue was azeotroped with toluene. The resulting oil wasdissolved in ethyl acetate, washed with water, 1 M sodium carbonate andbrine, dried over anhydrous magnesium sulphate and filtered. Thefiltrate was evaporated to leave a pale yellow oil which was purified byflash chromatography (silica gel, 10% to 25% ethyl acetate in hexane aseluant). Yield 3.43 g, 64%). ¹H-NMR; δ (CDCl₃, mixture ofα-diastereoisomers), 7.36–7.17 (10H, m), 5.80 (0.45H, br s), 5.55(0.55H, br d, J=8.9 Hz), 4.72–4.59 (3H, m), 4.20–4.05 (2H, m), 3.97(0.45H, m), 3.82 (0.55H, m), 3.47–3.22 (2H, m), 2.45 (1H, m), 1.90–1.48(2H, m), 1.40–1.14 (7H, m) and 0.95–0.84 (3H, m).

Step E: N-[2R-(4S-Benzyl-2-oxo-oxazolidine-3-carbonyl)-1R (orS)-methyl-hexyl]-N-benzyloxy-formamide

4S-Benzyl-3-[2R-(1R (orS)-benzyloxyamino-ethyl)-hexanoyl]-oxazolidin-2-one (3.08 g, 7.3 mmol)was dissolved in dry THF and treated with N-formylbenzotriazole (1.60 g,10.9 mmol). The reaction was stirred for 4 hours at room temperature.The solvent was removed under reduced pressure and the remaining oil waspartitioned between dichloromethane (40 ml) and 1 M sodium hydroxidesolution (30 ml). The organic layer was removed, washed with more sodiumhydroxide then brine, dried over anhydrous magnesium sulphate, filteredand evaporated. Purification by flash chromatography (silica gel, 20% to50% ethyl acetate in hexane) gave the title compound as a pale yellowsolid (2.50 g, 76%. ¹H-NMR; δ (CDCl₃, mixture of α-diastereoisomers androtamers), 8.22 (1H, br m), 7.54–7.13 (10H, m), 5.22–3.92 (7H, br m),3.30 (1H, m), 2.48 (1H, br m), 1.85–1.13 (9H, br m) and 0.93–0.83 (3H,m).

Step F: 2R-[1R (or S)-(Benzyloxy-formyl-amino)-ethyl]-hexanoic acid

N-[2R-(4S-Benzyl-2-oxo-oxazolidine-3-carbonyl)-1R (orS)-methyl-hexyl]-N-benzyloxy-formamide (1.50 g, 3.31 mmol) was dissolvedin THF (25 ml) and water (5 ml) and the solution was cooled in anice-water bath. Hydrogen peroxide solution (27% w/w), 13.26 mmol) wasadded followed immediately by lithium hydroxide (167 mg, 3.98 mmol). Thereaction was allowed to warm to room temperature and stirred for afurther 3 hours. The solution was cooled again before addition of sodiumnitrite (0.92 g, 13.3 mmol). After 10 minutes, most of the solvent wasremoved under reduced pressure to leave a white paste which waspartitioned between ethyl acetate (25 ml) and 1M sodium carbonate (30ml). The organic layer was washed with more sodium carbonate solutionand the combined aqueous extracts were washed with ethyl acetate. Theaqueous layer was cooled and acidified with 1 M hydrochloric acid andextracted twice with ethyl acetate.

The combined organic layers were washed with brine, dried over anhydrousmagnesium sulphate, filtered and evaporated to provide the titlecompound as a green oil (839 mg, 86%). ¹H-NMR; δ (CDCl₃, mixture ofα-diastereoisomers and rotamers), 8.40–7.64 (2H, br m), 7.48–7.27 (5H,m), 5.23–4.80 (2H, m), 4.16 (1H, br m), 2.79 (1H, m), 1.67–1.47 (2H, m),1.47–1.18 (7H, m) and 0.95–0.82 (3H, m).

Step G: 2R-[1R (or S)-(Benzyloxy-formyl-amino)-ethyl]-hexanoic acid(1S-dimethyl-carbamoyl-2,2-dimethyl-propyl)-amide

2R-[1R (or S)-(Benzyloxy-formyl-amino)-ethyl]-hexanoic acid (839 mg,2.86 mmol), tert-leucine N,N-dimethyl amide (498 mg, 3.15 mmol) and EDC(658 mg, 3.43 mmol) were dissolved together in DMF (15 ml) and acatalytic amount of HOAt (60 mg) was added. The solution was left tostir for several days at room temperature. The solvent was removed underreduced pressure and the remaining oil was partitioned between ethylacetate and 1 M hydrochloric acid (75 ml). The organic layer was washedsuccessively with 1 M hydrochloric acid, 1 M sodium carbonate and brine,dried over anhydrous magnesium sulphate filtered and evaporated to leavea yellow foam (1.08 g, 82%). ¹H-NMR; δ (CDCl₃, mixture ofα-diastereoisomers and rotamers), 8.13 (1H, br m), 7.52–7.31 (5H, m),6.28 (1H, br m), 5.36–4.67 (3H, br m), 4.09 (1H, br m), 3.14 (3H, s),2.95 (1.2H, s), 2.93 (1.8H, s), 2.48 (1H, br m), 1.61–1.04 (9H, m), 0.99(3.6H, s), 0.95 (5.4H, s) and 0.89–0.75 (3H, m).

Step H: 2R-[1R (or S)-(Formyl-hydroxy-amino)-ethyl]-hexanoic acid(1S-dimethyl-carbamoyl-2,2-dimethyl-propyl)-amide

2R-[1R (or S)-(Benzyloxy-formyl-amino)-ethyl]-hexanoic acid(1-dimethyl-carbamoyl-2,2-dimethyl-propyl)-amide (200 mg, 0.46 mmol) wasdissolved in methanol (15 ml) and placed under a blanket of argon. Asuspension of 10% palladium on charcoal (20 mg) in ethyl acetate wasadded and the mixture was stirred under an atmosphere of hydrogen for 3hours. The catalyst was removed by filtration and the filtrate wasevaporated to leave a colourless oil (163 mg, quant.). The twodiastereoisomeric products were separated by preparative HPLC.

Diastereoisomer A (27 mg): ¹H-NMR; δ (CDCl₃, mainly one rotamer), 8.67(0.9H, br s), 8.33 (0.1H, br s), 7.92 (1H, s), 6.74 (0.1H, br m), 6.54(0.9H, d, J=9.4 Hz), 4.93 (0.9H, d, J=9.4 Hz), 4.64 (0.1H, br m), 3.89(1H, qd, J=6.6, 2.6 Hz), 3.16 (3H, s), 2.96 (3H, s), 2.62–2.48 (1H, m),1.52–1.06 (6H, m), 1.35 (3H, d, J=6.6 Hz), 1.00 (9H, s) and 0.82 (3H, t,J=6.9 Hz). ¹³C-NMR; δ (CDCl₃), 173.0, 171.3, 57.2, 54.4, 50.4, 38.4,35.6, 29.9, 29.1, 26.6, 22.5, 17.2 and 13.9. LRMS: +ve ion 366 [M+Na],−ve ion 342 [M−H].

Diastereoisomer B (42 mg): ¹H-NMR; δ (CDCl₃, mixture of rotamers), 9.15(0.6H, s), 8.60 (0.4H, br s), 8.42 (0.6H, s), 7.84 (0.4H, s), 6.83(0.6H, d, J=9.2 Hz), 6.55 (0.4H, d, J=9.4 Hz), 4.91 (0.6H, d, J=9.2 Hz),4.89 (0.4H, d, J=9.4 Hz), 4.69 (0.6H, qd, J=7.0, 4.3 Hz), 3.92 (0.4H,dq, J=9.1, 6.8 Hz), 3.15 (3H, s), 2.97 (1.8H, s), 2.95 (1.2H, s), 2.59(0.4H, td, J=9.8, 4.3 Hz), 2.39 (0.6H, td, J=7.4, 4.3 Hz), 1.92–1.07(6H, m), 1.37 (1.2H, d, J=6.8 Hz), 1.31 (1.8H, d, J=7.0 Hz), 1.01 (5.4H,s), 0.96 (3.6H, s), 0.85 (1.8H, t, J=7.2 Hz) and 0.83 (1.2H, t, J=7.2Hz). ¹³C-NMR; δ (CDCl₃, mixture of rotamers), 175.7, 173.2, 171.3,170.7, 56.7, 55.0, 54.4, 53.2, 50.8, 49.9, 38.3, 35.7, 35.6, 35.5, 35.4,30.3, 29.5, 29.3, 26.5, 26.4, 22.5, 22.4, 16.0, 15.4 and 13.8. LRMS: +veion 366 [M+Na], −ve ion 342 [M−H].

EXAMPLE 70N-cyclohexyl-2-{2-[(formyl-hydroxy-amino)-methyl]-3-phenyl-propionylamino}-3,3-dimethyl-butyramide

Stock solutions of 1 M ammonia in methanol (1 ml, 1 mmol) and 1 Mtrimethylacetaldehyde in methanol (1 ml, 1 mmol) were mixed in a boilingtube and allowed to stand for 1 hour. A 1 M solution of cyclohexylisocyanide in methanol (1 ml, 1 mmol) was added followed by 0.5 M2RS-[(benzyloxy-formyl-amino)-methyl]-hexanoinc acid in methanol (2 ml,1 mmol). The reaction mixture was allowed to stir at room temperaturefor 2 days. The solvent was removed using a Savant Speedvac and thereaction mixture was crystallised from ethylacetate-hexane to provide2-{2-[(benzyloxy-formyl-amino)-methyl]-3-phenyl-propionylamino}-N-cyclohexyl-3,3-dimethyl-butyramideas a white solid (93 mg, 18%), which was deprotected by catalytictransfer hydrogenolysis (hydrogen gas, 10% palladium on charcoal,methanol-ethyl acetate) to provide the title compound (75 mg, 99%).White solid. LRMS: +ve ion 440 [M+Na], 418 [M+H], −ve ion 416 [M−H].

The compounds of Examples 71 to 77 were prepared in parallel using theUgi 4 component condensation reaction, as described above. All productswere obtained in >85% purity as determined by HPLC.

EXAMPLE 712-{2-[(Formyl-hydroxy-amino)-methyl]-3-phenyl-propionylamino}-3,3-dimethyl-hexanoicacid cyclohexyl amide

White solid (90 mg). ¹H-NMR; δ (CD₃OD), 7.82 (1H, s), 7.29–7.08 (5H, m),4.20 (1H, d, J=5.0 Hz), 3.89 (1H, m), 3.19 (1H, m), 2.95–2.67 (2H, m),1.88–1.58 (5H, br m), 1.44–1.05 (9H, br m) and 0.89 (9H, s). LRMS: +veion 468 [M+Na], 446 [M+H], −ve ion 444 [M−H].

EXAMPLE 722-{2-[(Formyl-hydroxy-amino)-methyl]-3-phenyl-propionylamino}-3,3-dimethyl-hexanoicacid phenylmethyl amide

White solid (77 mg). ¹H-NMR; δ (CD₃OD), 7.82 (1H, s), 7.35–7.11 (10H,m), 4.38–4.19 (3H, m), 3.85 (1H, m), 3.52 (1H, m), 2.97–2.63 (3H, m),1.37–1.11 (4H, m) and 0.93–0.78 (9H, m). LRMS: +ve ion 476 [M+Na], 454[M+H].

EXAMPLE 732-{2-[(Formyl-hydroxy-amino)-methyl]-3-phenyl-propionylamino}-3,3-dimethyl-butyricacid tert-butyl amide

White solid (47 mg). ¹H-NMR; δ (CD₃OD), 7.82 (1H, s), 7.45 (1H, m),7.30–7.09 (5H, m), 4.12 (1H, d, J=7.2 Hz), 3.89 (1H, m), 3.41 (1H, m),3.15 (1H, m), 2.97–2.68 (2H, m), 1.28 (9H, s) and 0.92 (9H, s). LRMS:+ve ion 414 [M+Na], 392 [M+H], −ve ion 390 [M−H].

EXAMPLE 742-{2-[(formyl-hydroxy-amino)-methyl]-3-phenyl-propionylamino}-3,3-dimethyl-hexanoicacid (1,1,3,3-tetramethyl)-butyramide

White solid (65 mg). ¹H-NMR; δ (CD₃OD), 7.79 (1H, s), 7.42–7.21 (1H, m),7.20–7.10 (5H, m), 4.23 (1H, d, J=9.1 Hz), 3.86 (1H, m), 3.51 (1H, m),3.23 (1H, m), 3.00–2.56 (2H, m), 1.50–1.15 (12H, m) and 1.02–0.83 (18H,m). LRMS: +ve ion 498 [M+Na], 476 [M+H, −ve ion 474 [M−H].

EXAMPLE 75N-(Cyclohexyl-cyclohexylcarbamoyl-methyl)-2-[(formyl-hydroxy-amino)-methyl]-3-phenyl-propionamide

White solid (98 mg). ¹H-NMR; δ (CD₃OD), 7.38–7.08 (5H, m), 4.01 (1H, m),3.81 (1H, m), 3.68–3.35 (2H, m), 3.15 (1H, m), 2.98–2.65 (2H, m),1.88–1.49 (10H, br m) and 1.45–0.83 (11H, br m). LRMS: +ve ion 466[M+Na], 444 [M+H], −ve ion 442 [M−H].

EXAMPLE 76N-(Cyclohexyl-phenylmethylcarbamoyl-methyl)-2-[(formyl-hydroxy-amino)-methyl]-3-phenyl-propionamide

White solid (34 mg). ¹H-NMR; δ (CD₃OD), 7.35–7.10 (10H, m), 4.44–4.23(2H, m), 4.05 (1H, m), 3.87–3.35 (2H, m), 3.09 (1H, m), 2.85–2.72 (2H,m), 1.65–1.46 (4H, m), 1.38–0.93 (5H, br m) and 0.75–0.51 (2H, br m).LRMS: +ve ion 474 [M+Na], −ve ion 450 [M−H].

EXAMPLE 77N-[Cyclohexyl-(1,1,3,3-tetramethyl-butylcarbamoyl)-methyl]-2-[(Formyl-hydroxy-amino)-methyl]-3-phenyl-propionamide

White solid (51 mg). ¹H-NMR; δ (CD₃OD), 7.80 (1H, s), 7.36–7.10 (5H, m),4.05 (1H, m), 3.85 (1H, m), 3.49 (1H, m), 3.15 (1H, m), 2.91 (1H, m),2.68 (1H, m), 1.90 (1H, m), 1.80–1.48 (7H, m), 1.40–1.12 (10H, m) and1.08–0.83 (10H, m). LRMS: +ve ion 496 [M+Na], 474 [M+H], −ve ion 472[M−H].

BIOLOGICAL EXAMPLE A Demonstration of Antibacterial Effect of Compound 1(Example 1) and Compound 2 (Example 13).

a).

Minimal inhibitory concentrations (MIC) of inhibitors against E. colistrain DH5α (Genotype; F-φ80dlacZΔM15Δ(lacZYA-argF)U169 deoR recA1 endA1hsdR17(r_(k) ⁻,m_(k) ⁺)phoA supE44λ⁻ thi-1 gyrA96 relA1) obtained fromGibcoBRL Life Technologies, Enterobacter cloacae (American Type CultureCollection number 13047), Klebsiella pneumoniae (American Type CultureCollection number 13883) or Staphylococcus capitis (American TypeCulture Collection number 35661) were determined as follows. Stocksolutions of test compound (Compounds 1 and 2 from Examples 1 and 2respectively (both isomer A)) and three standard laboratory antibiotics,carbenicillin (Sigma, catalogue No. C3416), kanamycin (Sigma, catalogueNo. K4000) and chloramphenicol (Sigma, catalogue No. C1919), wereprepared by dissolution of each compound in dimethylsulfoxide at 10 mM.For the determination of the minimal inhibitory concentration, two foldserial dilutions were prepared in 2×YT broth (typtone 16 g/1, yeastextract 10 g/1, sodium chloride 5 g/1 obtained from BIO 101 Inc, 1070Joshua Way, Vista, Calif. 92083, USA) to yield 0.05 mlcompound-containing medium per well. Inocula were prepared from culturesgrown overnight in 2×YT broth at 37° C. Cell densities were adjusted toabsorbance at 660 nm (A₆₆₀)=0.1; the optical density-standardizedpreparations were diluted 1:1000 in 2×YT broth; and each well inoculatedwith 0.05 ml of the diluted bacteria. Microtiter plates were incubatedat 37° C. for 18 hours in a humidified incubator. The MIC (μM) wasrecorded as the lowest drug concentration that inhibited visible growth.

TABLE 1 MIC μM carbenicillin chloramphenicol kanamycin compound 1compound 2 E. coli DH5α 25 3.12 12.5 12.5 6.25 Staphylococcus <1.56 6.25<1.56 100 25 capitis Enterobacter >200 25 50 50 25 cloacae Klebsiella200 12.5 25 25 12.5 pneumoniaeb).

Minimal inhibitory concentrations (MIC) of inhibitors againstMycobacterium ranae (American Type Culture Collection number 110),Pseudomonas aeruginosa (American Type Culture Collection number 9027),Klebsiella pneumoniae (American Type Culture Collection number 10031),Helicobacter pylon (American Type Culture Collection number 43504),clinical isolates of aminoglycoside and erythromycin resistantStreptococcus pneumoniae and methicillin-resistant (MR) Staphylococcusaureus (American Type Culture Collection number 33591) were determinedas follows. Stock solutions of test compounds 1 and 2 (isomer A foreach) and three standard laboratory antibiotics, gentamycin (G),ampicillin (A) and erythromycin (E), were prepared by dissolution ofeach compound at 10 mg/ml in dimethylsulfoxide. Methods used were as fora) except that the medium of Mycobacterium ranae was used with BrainHeart Infusion broth (GIBCO) and incubated at 37° C. for 48 hours,Staphylococcus aureus (MR), Klebsiella pneumoniae, and Pseudomonasaeruginosa were used with Nutrient Broth (DIFCO) and incubated at 37° C.for 20 hours, Helicobacter pylori was used with Columbia agar base(OXOID) containing 7% sheep blood and incubated at 35° C. for 72 hoursand, Streptococcus pneumoniae was used with tryptic soy broth (DIFCO)containing 7% calf serum and incubated at 37° C. for 48 hours. The MIC(μg/ml) was recorded as the lowest drug concentration that inhibitedvisible growth.

Positive vehicle control (1% DMSO; no test agent) caused growth of allmicroorganisms.

Negative blank control (absence of microorganisms; +test agent) revealedno growth of microorganisms.

TABLE 2 MIC μg/ml antibiotic compound 1. compound 2. G A E M. ranae 0.780.2 0.2 nd nd S. aureus (MR) 3.13 1.56 0.78 nd nd K. pneumoniae 0.2 0.10.39 nd nd P. aeruginosa 12.5 12.5 0.78 100 nd H. pylori 0.1 0.1 0.780.1 nd S. pneumoniae 50 12.5 100 3.13 100 nd = not determined.

In another experiment, minimal inhibitory concentrations of compounds 1and the product of Example 13 (compound 3) against a range ofGram-positive and Gram-negative bacteria were determined using theMicrodilution Broth Method according to the approved standard of theNational Committee for Clinical Laboratory Standards procedure (Methidsfor dilution antimicrobial susceptibility tests for bacteria that growaerobically-Fourth Edition ISBN 1-56238-309-4)

Activity against Gram-positive bacteria MIC μg/ml Bacterial StrainCompound 3 Compound 1 Vancomycin Staphylococcus aureus 8 32 0.25 ATCC29213 MSSA Staphylococcus aureus 16 16 0.5 ATCC 25923 MSSAStaphylococcus aureus 4 8 0.5 ATCC 6538 MSSA Staphylococcus epidermidis4 8 0.5 ATCC 1228 Staphylococcus epidermidis 2 8 0.5 ATCC 27626Enterococcus faecalis 32 32 1 ATCC 29212 Enterococcus faecalis 8128 >128 (Vancomycin resistant strain) Micrococcus luteus 0.5 0.5 0.25ATCC 9341

Activity against Gram-negative bacteria MIC μg/ml Bacterial StrainCompound 3 compound 1 Ciprofloxacin Escherichia coli 4 4 <0.125 ATCC25922 Escherichia coli 4 4 <0.125 ATCC 12014 Pseudomonas aeruginosa128 >128 <0.125 ATCC 27853 Enterobacter cloacae 32 32 <0.125 ATCC 13047Morganella morganii >128 128 <0.125 ATCC 36030 Klebsiella pneumoniae 1616 <0.125 ATCC 13883

The activities of compound 3 and the product of Example 14 (compound 4)against a multi-resistant Enterococcus faecalis clinical isolate,assessed by the method used for the immediately preceding results, areset out in the following table, and compared with the results obtainedby the same method for known antibacterial agents:

MIC μg/ml Bacterial Strain Cpd 3 Cpd 4 Ampicillin Ceftazidime ImipenemErythromycin Ciprofloxacin Vancomycin Enterococcus 32 8 0.5 128 1 2 0.51 faecalis ATCC 29212 Enterococcus 8 4 >128 >128 >128 >128 >128 >128faecalis Vancomycin resistant strain

BIOLOGICAL EXAMPLE B

i) Cloning of the Escherichia coli PDF Gene.

The E. coli PDF gene was cloned in pET24a(+) (designated pET24-PDF) andwas used to transform BL21 DE3 cells from Novagen Inc, (Madison, Wis.).Clones were selected at 37° C. on YT agar plates (8 g/l typtone, 5g/yeast extract, NaCl 5 g/l, agar 15 g/l) supplemented with 30 μg/mlkanamycin.

ii) Expression of PDF

A 20 ml overnight culture of BL21 DE3 cells harbouring pET24-PDF wasused to infect 500 ml 2×YT broth (16 g/l typtone, 10 g/l yeast extract,NaCl 5 g/l) containing 30 ug/ml kanamycin in a 2 liter baffled flask andgrown at 37° C. with shaking to an OD₆₀₀ 0.6. The culture was theninduced by adjusting the medium to 1.0 mM isopropyl β-Dthiogalactopyranoside (IPTG). The induction was allowed to proceed for afurther 3 hours at 37° C., the cells were harvested by centrifugationand the cell pellet washed with 250 ml phosphate buffered saline (PBS)and the pellet stored at −70° C.

iii) Preparation of Soluble Protein Fraction.

The cells from a 1 liter expression were resuspeneded in 2×25 ml of icecold phosphate buffered saline. The cell suspension was sonicated on iceusing an MSE Soniprep 150 fitted with a medium probe and at an amplitudeof 20–25 microns in 6×20 second pluses. The resulting suspension wasthen cleared by centrifugation at 20,000×g for 15 minutes. Thesupernatant was then used for further purification of the enzyme.

iv) PDF Purification

E. coli lysate from a 1 l culture in phosphate buffered saline (PBS)were adjusted to 2M ammonium sulphate. A 15 ml phenyl sepharose columnwas equilibrated with PBS/2M ammonium sulphate at 4° C. The lysate wasloaded on the column and washed with equilibration buffer. The columnwas eluted by reducing the ammonium sulphate concentration from 2M to 0Mover 10 column volumes. 5 ml fractions were collected and analysed bySDS-PAGE. The fractions containing the majority of the 20 kDa PDF werepooled. The pooled fractions were concentrated using a 3 kDa cutoffmembrane to a volume of 5 ml. The fraction was then loaded onto aSuperdex 75 (size exclusion chromatography) column equilibrated in PBS.The concentrated PDF pool eluted at one m/min at 4° C. and 5 mlfractions collected and analysed by SDS-PAGE. The purest fractions werepooled and stored at −70° C.

(v) PDF In Vitro Assay

The assay was performed in a single 96 well plate in a final volume of100 μl containing:

-   -   20 μl PDF (4 μg/ml)    -   20 μl 100 mM Hepes pH 7.0+1M KCl+0.05% Brij    -   10 μl serial dilution of test compound in 20% DMSO    -   50 μl formyl-Met-Ala-Ser (8 mM)

The assay was incubated at 37° C. for 30 minutes. The free amino groupof the deformylated (Met-Ala-Ser) product was detected usingfluorescamine, by the following additions:

-   -   50 μl 0.2M borate pH 9.5    -   50 μl fluorescamine (150 μg/ml in dry dioxane)

Fluorescence was quantified on SLT Fluostar plate reader using anexcitation wavelength of 390 nM and an emission wavelength of 485 nM.Standard control reactions are a no inhibitor reaction which providesthe zero inhibition figure and a no enzyme and no inhibitor reactionwhich provides the 100% inhibition figure. The data was analysed byconversion of the fluorescence units to % inhibition and the inhibitorconcentration plotted against % inhibition. The data was fitted to asigmoidal function: y=A+((B−A)/(1+((c/x)^(D)))), wherein A representszero inhibition, B represents 100% inhibition and C represents the IC₅₀,D represents the slope. The IC₅₀ represents the concentration ofinhibitor (nM) required to decrease enzyme activity by 50%.

Compounds of the invention were found to inhibit bacterial PDF in vitro.In addition, actinonin (Sigma Cat. No. A-6671) was also found to inhibitbacterial PDF in vitro.

BIOLOGICAL EXAMPLE C

Demonstration that Compound 2 Inhibits PDF In Vivo.

1 Blocking the tRNAi-Met Transformylation Reaction Confers Resistance toCompound 2 (Diastereomer/Isomer A).

Trimethoprim specifically inhibits dihydrofolate reductase, therebydepressing the pools of tetrahydrofolate (THF) derivatives, includingformyl tetrahydrofolate (fTHF), the substrate of the methionyl-tRNAformyltransferase (EC 2.1.2.9). If all essential metabolites whosebiosynthesis involves THF derivatives, eg pantothenate, methionine,glycine, purine nucleotides and thymidine are supplied exogenously inthe form of precursor compounds in rich medium supplemented withthymidine, then bacteria grown in rich medium plus thymidine (0.3 mM)and trimethoprim (100 μg/ml) can synthesize all the chemical componentsof normal cells except f-Met-tRNAi (Baumstark et al., J. Bacteriol.129:457–471, 1977). Unformylated Met-tRNAi is used instead, resulting inthe formation of polypeptides devoid of a formyl group at theirN-terminus, independently of the action of deformylase. As predicted bythe inventors, DH5α cells grown in LB medium (typtone 10 g/l, yeastextract 5 g/l NaCl 10 g/l pH7.5) supplemented with trimethoprim andthymidine, were found to be resistant to compound 2 (diastereomer A).The demonstration that cells that undergo the normal formylation processon expressed proteins are inhibited by compound 2A whereas, unformylatedproteins, as produced in the cells grown under these conditions, are notinhibited by compound 2A demonstrates that compound 2A is likely to workby inhibiting the deformylation reaction carried out by PDF.

TABLE 3 minimal inhibitory growth conditions concentration μM LB 15 LBtrimethoprim (100 μg/ml), >200 thymidine (0.3 mM)2 Treatment of Bacteria with Compound 2A Leads to the Accumulation ofN-Terminally Blocked Proteins.

If the compounds of the invention do actually inhibit PDF in vivo, thena consequence of treatment of bacteria with compound 2 (Example 2,diastereomer A) will be the accumulation of N-formyl methionine at theN-terminus of newly synthesised proteins. Such proteins will beN-terminally blocked and will be unable to be used as a substrate forN-terminal sequencing by Edman degradation chemistry.

To test this hypothesis a desired protein is expressed in the presenceor absence of the test compound. The protein is isolated, purified andthen subjected to Edman degradation protein sequencing using techniquesknown to the person skilled in the art.

Bacterial cells transformed with an expression vector allowingexpression of the human calpain small regulatory subunit, were grown toan OD₆₀₀ of 0.6 and then subjected to IPTG to induce expression of theheterologous protein in the presence of 200 μM compound 2A, in thepresence of 240 μM carbenicillin or, in the presence of vehicle controlfor 2.5 hours. Protein extracts were separated by SDS-PAGE, the calpainsubunit eluted and the protein sequence determined by Edman degradationchemistry using the ABI automated protein sequencer. Equal quantities ofprotein were sequenced. The inventors found that the yield of thecompound 2A treated protein was significantly reduced by 85% compared tovehicle and carbenicillin treated controls.

Calpain small regulatory subunit was cloned from messenger RNA obtainedfrom a gastric tumour biopsy using the InVitrogen Micro Fast Track™ mRNAisolation kit version 2.2 (catalogue number K1520–02). Copy DNA fromthis mRNA was synthesised using the Promega Riboclone™ cDNA synthesissystem M-MLV-RT(H-), NotI (Promega, Catalogue number C1660) according tothe manufactures instructions. Two oligonucleotide primers for use inthe polymerase chain reaction (PCR) were synthesised by AppliedBiosystems, Inc., Custom Services, based on the published calpain smallsubunit sequence (EMBL Accession number X04106).

The HindIII/XhoI calpain fragment was then cloned into HindIII and XhoIdigested expression vector pET24d(+) from Novagen Inc, (Madison, Wis.,USA) using standard procedures. The ligation mixture was used totransform competent DH5αcells (Life Technologies, Inc, Grand Island,N.Y., USACat# 18265–017). Colonies were selected by growth overnight at37° C. on YT plates plus 30 μg/ml kanamycin. Plasmid DNA was prepared asusing the Promega Plus SV miniprep kit and clones with the calpaininsert were identified using standard procedures. The DNA sequence wasconfirmed using the PE Applied Biosystems cycle sequencing as describedabove.

The E. coli gene cloned in pET24d(+) (designated pET24-CANS) was used totransform BL21 DE3 cells from Novagen Inc, (Madison, Wis.). Clones wereselected at 37° C. on YT agar plates (8 g/l typtone, 5 g/yeast extract,NaCl 5 g/l, agar 15 g/l) supplemented with 30 μg/ml kanamycin.

1. A compound of formula (I) or a pharmaceutically or veterinarilyacceptable salt thereof:

wherein: A represents a group of formula (IA) or (IB)

and wherein: R₁ is hydrogen, R₂ is n-butyl, benzyl or cyclopentylmethyl,R₃ is hydrogen, R₄ is tert-butyl, iso-butyl, benzyl or methyl, R₅ ishydrogen or methyl, and R₆ is methyl.
 2. A compound as claimed in claim1 selected from 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(1S-dimethylcarbamoyl-ethyl)-amide or a pharmaceutically or veterinarilyacceptable salt thereof.
 3. A compound as claimed in claim 1 selectedfrom 2R (or S)-[(Formyl-hydroxy-amino)-methyl]-3-cyclopentyl-propionicacid (1S-dimethyl-carbamoyl-2,2-dimethyl-propyl)-amide or apharmaceutically or veterinarily acceptable salt thereof.
 4. Anantibacterial pharmaceutical or veterinary composition comprising acompound as claimed in claim 1 together with a pharmaceutically orveterinarily acceptable excipient or carrier.